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

Satu Hepojoki,Jussi Hepojoki,Antti Vaheri,Olli Vapalahti,Visa Nurmi,Klaus Hedman,Sabato D'Auria

doi:10.1371/journal.pone.0106432

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.

Highlights

Biological sample materials are prone to autofluorescence, which can be minimized by utilizing time-resolved fluorometry (TRF)
Since TR-Forster resonance energy transfer (FRET) signal is proportional to the distance between donor and acceptor, we explored the possibility of using a generic Fab-binding molecule and a labeled antigen as constituents of a Time-resolved FRET (TR-FRET) -based homogeneous immunoassay
The results indicate that increasing the concentration of protein L in the reaction mixture in proportion to IgG produces higher TR-FRET signals, and increases background resulting in lower signal to noise (S/N) ratios

Summary

Introduction

Biological sample materials are prone to autofluorescence, which can be minimized by utilizing time-resolved fluorometry (TRF). TRF takes advantage of unique rare earth elements called lanthanides, such as europium, which have exceptionally long fluorescence emission half-lives. In Forster resonance energy transfer (FRET), energy is transferred between two fluorophores, the donor and the acceptor [1]. Time-resolved FRET (TR-FRET) unites the properties of TRF and FRET, which is especially advantageous when analyzing biological samples. As TR-FRET -based methods induce relatively low background fluorescence, this technique has been widely applied in medical research and diagnostics [2,3,4,5,6,7,8,9,10]. TR-FRET -based applications offer a viable alternative for the conventional multistep diagnostic tests, such as enzyme-linked immunosorbent assay (ELISA)

Methods

Results

Conclusion