Rapid antigen assays are very important for tracking the spread of an infectious disease during a pandemic. Though the rapid assays for SARS-CoV-2 offer benefits over real-time RT-PCR assays but lag behind in terms of sensitivity (Funabashi et al., 2021). Most of the rapid antigen assays were designed using N protein as it is preferred because of its relative abundance (Juniastuti et al., 2023) and is relatively stable even in variants (Mohammad et al., 2021) while very few assays are developed using the S protein as target for screening of SARS-CoV-2. As per 2022 data, among the 44 FDA-EUA antigen-detecting lateral flow assays, 42 are directed against the N antigen whereas only two target both N antigen and receptor binding domain (RBD) of the S protein (Ang et al., 2022). In one of the studies, the antigen assay developed using N protein has been able to detect all samples with high and medium-viral titers, while it could detect 64.7% (95% CI: 47.8% − 78.6%) samples in the low-virus titer cohort, but the assay is negative for PCR negatives (Funabashi et al., 2021). Even ELISA assays developed using mAbs against N protein revealed a sensitivity and specificity of 70.72% (95% CI: 66.01–75.12) and 100% (95% CI: 97.57–100), respectively, regardless of Ct values and SARS-CoV-2 variants (Yadegari et al., 2023). Another study showed a clinical sensitivity and specificity of 85.2% (95% CI, 74.3–92.0%) and 98.1% (95% CI, 93.3–99.7%) respectively against real-time PCR (Fischl et al., 2024).
Tests using commercial rapid antigen assays revealed a sensitivity ranging from 60.55 to 87.23% with high specificity ranging between 83.33 and 100% in all tests in samples with Ct value < 20 against the real-time PCR assay in case of Delta variants (Samsunder et al., 2023a). Similarly, while screening samples infected with Omicron sub-lineage BA.4 and BA.5, sensitivities of 73.38–74.03% and specificities of 99.22–97.41% respectively were reported using two commercial kits. Sensitivity was reported > 90% when the Ct value was < 20 (Samsunder et al. 2023b). In one of the studies where the sensitivities of rapid antigen assays were evaluated against the mutant variants, it was found that most commercially available rapid antigen tests (RATs) had similar sensitivity in detecting Omicron and Delta variants. When the antigen concentration was used as a comparator and the Ct value was used as a comparator, most rapid assays had a lower sensitivity for Omicron than Delta (Rao et al., 2023). A nanoparticle based lateral flow assay employing N protein in comparison to the RT-PCR, showed a sensitivity of 94.73% (Ding et al., 2023).
In one of the studies on performance evaluation of six commercial kits, each test showed no difference in the detection sensitivities between the wild-type virus and the variants thus suggesting that the lateral flow antigen test can be used for the detection of SARS-CoV-2 like the wild-type and the previous variants (Morinaga et al., 2023). However, some studies report regarding the reduced sensitivity of antigen tests for screening the Omicron variant in comparison with the previous variants or the wild-type virus (Osterman et al., 2022; Wagenhauser et al., 2023). As per WHO recommendations, the acceptable criteria for comparison of a rapid assay with the reference method i.e., RT-PCR needs 80% sensitivity and 97% specificity (Dinnes et al., 2022; WHO, 2021), while the in-house developed assay in the present study meets these standards with 99.05% sensitivity and 100% specificity. Further, with the periodic surges of COVID-19 globally and the continuous spread of infections emphasise the importance of rapid diagnostics. In addition, there is an inherent need to make a data repository of signature motifs from these aforementioned assays which can then be established using immunoprecipitation analysis with biotinylated transcripts. These in turn could be potential targets for designing the aptamers which would ideally be specific and then test their efficacy for aptamer bound lateral flow assay towards theranostic validation.
Hence, there is the need for reliable antigen based rapid detection assays which are invaluable for the timely detection of SARS-CoV-2 infection with subsequent contact tracing and rapid isolation. However, there are some limitations to the present study, as the study population includes only symptomatic individuals. In addition, the screening of the new variant JN.1 was not done, but the developed assay may detect that too. Overall, the present study’s findings showed that the in-house developed COVID-19 rapid antigen test can exhibit excellent diagnostic performance and analytical sensitivity in detecting variants, including Omicron as the epitopes selected for S and N are derived from the regions where there are the least chances of mutations.
In response to COVID-19 infection, diagnostic real-time RT-PCR tests are adopted as a gold-standard method for detecting virus antigens and identifying COVID-19 patients as the method is more sensitive than rapid antigen assays. However, the real-time RT-PCR is time-consuming, and requires equipment that cannot be used under POC settings. Antigen-based rapid assays play an invaluable role in the rapid identification of highly infectious cases, which can generally provide rapid results without the need for complex instrumentation and technical expertise.