A Generic, Scalable, And Rapid Time-Resolved Forster Resonance Energy Transfer-Based Assay For Antigen Detection - Sars-Cov-2 As A Proof Of Concept

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 detect-ing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in na-sopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleo-protein (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 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) nasopharyn-geal 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 infec-tions. While PCR and its variants are highly sensitive and specific, the time from sam-pling 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 sam-ples 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 vi-rus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of con-cept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.