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Rapid Homogeneous Immunoassay Based on Time-Resolved Förster Resonance Energy Transfer for Serodiagnosis of Acute Hantavirus Infection

ABSTRACT We recently introduced a homogeneous immunoassay based on time-resolved Förster resonance energy transfer (TR-FRET) elicited by fluorophore-labeled antigen and fluorophore-labeled protein L, bound by an immunoglobulin. As the first clinical application, we employ this approach (LFRET) in serodiagnosis of Puumala hantavirus (PUUV) infection. A reference panel containing serum from individuals with acute (n = 21) or past (n = 17) PUUV infection and from PUUV-seronegative individuals (n = 20) was used to define the parameters. The clinical assay performance was evaluated with a prospectively collected serum panel (panel 2; n = 153). Based on the results for panel 1, the threshold for positivity was set at a signal level that was 3-fold over background, while those with a signal <3-fold over the background level were considered PUUV seronegative. With panel 1, 20/21 acute- and 7/10 past-infection samples induced positive signals, compared to 0/20 seronegatives. With panel 2, a positive signal was obtained in 39/40 acute- and 4/10 past-infection samples, as opposed to 7/103 seronegatives. However, after IgG depletion, 58/61 acute-infection samples were LFRET positive, while all past-infection and seronegative samples were negative, corresponding to 100% specificity and 95% sensitivity in detection of acute PUUV infection. We demonstrate that the novel immunoassay is a promising tool for rapid serodiagnosis of acute Puumala virus infection.