Search

The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤ 35 and ≤40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples.