Time-resolved Förster resonance energy transfer for rapid infectious disease serodiagnosis

Satu Hepojoki

Tutkimustuotos: OpinnäyteVäitöskirjaArtikkelikokoelma


Novel molecular tools for infectious disease diagnostics are constantly under development to reduce the time between onset of symptoms and diagnosis. Not only is it important to receive appropriate treatment, but also to avoid unnecessary use of antibiotics. The availability of rapid diagnostics is also important when epidemics or pandemics emerge. The purpose of this project was to examine the applicability of Förster resonance energy transfer (FRET) in homogeneous immunoassays, and to develop new diagnostic approaches. FRET has widely been applied in proximity-based assays, such as those measuring antigen-antibody binding. In FRET, energy is transferred between two chromophores, the donor and the acceptor, when in close proximity. Utilizing FRET as detection method for immunoassays enables the development of wash-free (homogeneous) simple workflow assays. In this thesis, of the three FRET-based rapid immunoassays that were set up, two served in clinical diagnosis. Study I (I) examined the possibility of detecting antibodies by FRET-pair forming fluorophore-labeled antigens, which upon binding to an antibody would induce a FRET signal. This homogeneous immunoassay, designated FRET-bridge, was successfully optimized for streptavidin (SA). By combining donor-labeled and acceptor-labeled SAs with anti-SA antibodies, FRET signals were recorded with high signal-to-noise ratios. When molecular determinants behind the FRET signals were examined, most of the FRET activity originated from fairly large immunocomplexes rather than from one IgG and two antigens. At the moment, SA represents the only antigen fully functioning in the FRET-bridge assay. This is most likely due to the multivalent nature of the antigen, which seems beneficial in FRET signal formation. Next, we introduced another homogeneous immunoassay (II), the LFRET assay. Here, a FRET pair forming fluorophore-labeled antigen and fluorophore-labeled protein L induce a signal if bound to the Fab (fragment antigen-binding) region of an immunoglobulin. To demonstrate the usefulness of the assay, SA served as test antigen. The assay was next optimized for virus diagnostics by use of Puumala virus (PUUV) nucleocapsid protein as antigen (III). In all, 211 serum samples underwent examination by the LFRET assay, representing acute (n=61) or past PUUV infection (n=27), and seronegative (n=123) individuals. With a simple workflow and an assay time of 30 minutes, the LFRET assay, compared to the reference tests, identified acute PUUV infection at 100% specificity and 95% sensitivity. The fourth study (IV) involved a competitive homogeneous immunoassay for the detection of PUUV antibodies from clinical samples. This assay, CFRET, is based on competition between fluorophore-labeled monoclonal antibodies (MAbs) and serum antibodies. Here, a donor-labeled antigen and an acceptor-labeled MAb form the FRET pair. If the clinical sample contains antibodies against the labeled antigen, they compete with the MAb for antigen binding, resulting in FRET signal decrease. Analysis of assay performance included a panel of 329 samples representing acute (n=101) or past (n=42) infection, and negative samples (n=186). The one-step CFRET assay performed at 99% specificity and 100% sensitivity in diagnosis of hantavirus disease compared to the reference tests, and with a rapid assay time of 30 minutes. The three rapid diagnostic approaches introduced herein represent simplicity, and show that diagnostics need not be time-consuming. Although the assays were optimized for accurate diagnosis of acute infection, both assays also recognized life-long immunity, albeit with lower sensitivity. By optimization, the assays could be developed towards more sensitive detection of past infection as well. The LFRET and CFRET assays thus represent innovative tools for rapid antibody detection, and their potential in serodiagnosis of diverse microbial infections and possibly even in detection of auto- and anti-allergen antibodies calls for further exploration.
Painoksen ISBN978-951-51-2477-7
Sähköinen ISBN978-951-51-2478-4
TilaJulkaistu - 2016
OKM-julkaisutyyppiG5 Tohtorinväitöskirja (artikkeli)


M1 - 72 s. + liitteet
Helsingin yliopisto


  • Antibodies
  • +analysis
  • Antibodies, Bacterial
  • +immunology
  • Antibodies, Viral
  • +blood
  • Bacterial Proteins
  • Enzyme-Linked Immunosorbent Assay
  • DNA-Binding Proteins
  • Fluorescence Resonance Energy Transfer
  • +methods
  • Hantavirus
  • Hantavirus Infections
  • +diagnosis
  • Immunoassay
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • Immunologic Tests
  • Limit of Detection
  • Protein Binding
  • Puumala virus
  • Sensitivity and Specificity
  • Serologic Tests
  • Time Factors
  • 3111 Biolääketieteet

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