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Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

ABSTRACT: Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.

SUBMITTER: Saraheimo S

PROVIDER: S-EPMC3647052 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

Saraheimo Satu S Hepojoki Jussi J Hepojoki Jussi J Nurmi Visa V Lahtinen Anne A Hemmilä Ilkka I Vaheri Antti A Vapalahti Olli O Hedman Klaus K

PloS one 20130507 5

Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule i ...[more]

PMID: 23667515

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Project description:The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.

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Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

Publications

Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

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