A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept

Abstract

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 [C_T ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with C_T 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.

Keywords: COVID-19; SARS-CoV-2; TR-FRET; antigen test; mix and read; rapid diagnostic test.

FIG 1

The TR-FRET assay workflow and principle. The left side shows the reaction components. At the center is a well containing donor- and acceptor-labeled antibodies at a 1:1 molar ratio in the reaction buffer; in our setup, the total antibody concentration at this point is 24 nM, and the volume is 10 μl. The arrows indicate the addition of the sample material, in our setup either 10 μl of purified recombinant protein or 10 μl of an NPS sample. The top right side shows schematically the antigen-antibody complexes formed following the addition of a sample containing the antigen; the reaction volume at this stage in our setup is 20 μl. The bottom right side schematically demonstrates that the labeled antibodies do not form TR-FRET active complexes in the absence of the antigen.

FIG 2

Comparison of time-resolved Förster resonance energy transfer (TR-FRET)-based antigen detection and the amount of virus as analyzed by SARS-CoV-2 RT-PCR and virus isolation from NPS samples. The total antibody concentration in the assay mixtures is 12 nM (6 nM Eu- and 6 nM AF647-labeled antibodies). (a) Anti-NP (nucleoprotein) assay results. (b) Anti-RBD (receptor-binding domain) assay results. The y axis (log scale) indicates the fold increase in the homogeneous time-resolved fluorescence (HTRF) ratio (HTRF_sample/HTRF_buffer) measured directly after pipetting the samples onto the plate. The x axis shows the C_T values measured in the diagnostic SARS-CoV-2 RT-PCR. The horizontal black line is the antigen test positivity cutoff, corresponding to the average plus 4 standard deviations of the signals induced by SARS-CoV-2 RT-PCR-negative samples. The vertical black line separates SARS-CoV-2 RT-PCR-positive (n = 48) and -negative (n = 96) NPS samples. The coloring in the graphs indicates the presence (red) or absence (blue) of cytopathic effect (CPE) following inoculation of VE6-TMPRSS2-H10 cells with 50 μl of the NPS sample. Black, not cultured.

FIG 3

SARS-CoV-2 TR-FRET antigen assay cross-reactivity evaluated with cell culture supernatants of seasonal human coronaviruses hCoV-229E and -NL63. (a) Anti-NP assay results with hCoV-229E and -NL63 cell culture supernatants at different dilutions. (b) Anti-NP assay results with hCoV-229E and -NL63 cell culture supernatants at different dilutions. The y axis (log scale) indicates the fold increase in the HTRF ratio (HTRF_sample/HTRF_buffer). The horizontal lines indicate the respective TR-FRET assay cutoffs. A UV-inactivated SARS-CoV-2-containing cell culture supernatant is included as a positive control.