Abstract
ABSTRACTThe 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.
Highlights
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 tested the assay principle by mixing recombinant NP and S protein (SP) with 1 mM (500 nM Eu-labeled and 500 nM Alexa Fluor 647 (AF647)-labeled) anti-NP and anti-receptor-binding domain (RBD) antibody mixtures in the presence of increasing bovine serum albumin (BSA) concentrations
While RTPCR is very sensitive in picking up individuals with acute infection, the downside is that patients recovering from COVID-19 can remain reverse transcription-PCR (RT-PCR) positive over a long period
Summary
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. 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. 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. We describe a technique for rapid antigen detection and demonstrate the test format’s potential using SARS-CoV-2 as the model pathogen. Detection of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or its parts is the cornerstone of diagnosis, as the disease presentation is often indistinguishable from those of other respiratory infections. SARS-CoV-2 is an enveloped positive-sense single-stranded RNA [(1)ssRNA] virus of the genus Betacoronavirus subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales Chelated lanthanide donor-enabled TR-FRET allows measurement from autofluorescent biological samples
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