RESUMO
The ongoing 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 method for the detection of SARS-CoV-2 in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Forster 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 viral units (i.u.), and 875 pg of SP or 625 i.u. of SARS-CoV-2. Testing RT-PCR positive (n=48, with cycle threshold [Ct] values from 11 to 30) or negative (n=96) nasopharyngeal swabs, we showed that the assay yields positive results for all samples with Ct values of <25 and a single RT-PCR negative sample. We determined the presence of infectious virus in the RT-PCR-positive nasopharyngeal swabs by virus isolation, and observed 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) and 99.0% (95/96) in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity and is relatively simple to set up as an "in-house" test. The assay principle as such is applicable to other viral infections, and could also be readily adapted to a massively high throughput testing format.
RESUMO
Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and will help to examine possible vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (S) and nucleocapsid protein (N). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Forster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal generation. We set up LFRET assays for antibodies against S and N and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described S construct and a novel N construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 S and N. We then compared the LFRET assays with these enzyme immunoassays and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90-95% vs. 90-100%) and specificity (100% vs. 94-100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91-96% vs. 82-87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-minute mix and read assay, to detection of SARS-CoV-2 antibodies.
