SARS-coV-2 IgM and IgG antibodies detection by ELISA.
Here, we analyzed the relationship between the levels of serum antibodies and time since symptoms onset. A total of 46 samples were collected from RT-PCR confirmed COVID-19 patients at several time points ranging from 3 to 27 days post symptoms onset, and the levels of SARS-CoV-2 specific IgM and IgG antibody responses were determined using in-house ELISA that we recently developed and validated. As shown in Figure 1A, the level of IgM antibodies started to increase by the end of the first week and reached their highest levels at day 11 before dropping down to lower levels which were maintained above the initial levels until day 27. Similarly, IgG levels started to elevate by day 7 and peaked by day 10 to levels that were remained high until day 27 post symptoms onset.
The ELISA median optical density (OD) of the negative samples was 0.4 (ranging from 0.03 to 0.53) for IgM and 0.14 (ranging from 0.1 to 0.2) for IgG. On the other hand, the median OD values of the 46 RT-PCR confirmed positive cases were significantly higher for both IgM (0.82; ranging from 0.08 to 4.86) and IgG (2.75; ranging from 0.07 to 4.16). Based on the predetermined cut-off values (Algaissi et al., 2020), and the samples used in this study, the overall sensitivity of IgG ELISA vs RT-PCR was 83% (95% CI: 63-91%; 38/46). All the 8 false negative samples were from samples collected at early time points post symptoms onset (6 samples during the first week and 2 samples on day 8). Thus, the IgG positivity in samples collected at late time points post symptoms onset was detected in 33/35 of the RT-PCR confirmed cases resulting in 94% sensitivity (95% CI: 81-99%). Importantly, all samples collected post day 8 were IgG positive, confirming our previous high sensitivity of IgG ELISA based on N protein (Algaissi et al., 2020). As expected, lower overall sensitivity of 75% was observed for the IgM ELISA (95% CI 83-63%; 30/46) compared to IgG ELISA, with 16 false negative samples divided between early (9 samples during the first week) and late time points (7 samples between days 8 and 11) post symptoms (Figure 1B). Nevertheless, all samples that were negative for IgG antibodies were IgM negative as well.
Detection of SARS-CoV-2 specific antibodies by ELISA and LFIA vs RT-PCR.
Next, we compared the performance of seven LFIA devices as well as our in-house ELISA with RT-PCR results, considering the levels of serum antibodies at early and late time-points post symptom onset (Figure 1), and the difference in measured targets by serological assays and RT-PCR. Therefore, any LFIA and ELISA positive results (IgG, IgM or both) were considered positive, and results were divided into two sets based on the peaking time points post symptoms onset (set1: one week and set2: after one week). To this end, the sensitivity of the seven tested LFIA devices and the ELISA compared to RT-PCR positive cases for set1 was very low ranging from 0% (95% CI 0-49%) to 54% (95% CI 28-79%) (Figure 2A). However, the sensitivity of the seven LFIA devices was increased to range from 54% (95%CI 38-69%) to 88% (95% CI 73-95%) for set2 (Figure 2B). This increase was also observed with the ELISA achieving 94% (95% CI 81-99%) sensitivity (Figure 2B). Moreover, the overall specificity of the seven LFIA devices and the ELISA was 100% (95% CI 80-100%) (Figure 2C).
Detection of SARS-coV-2 antibodies by ELISA vs LFIA
Having demonstrated differences in the sensitivity between early and late time points post symptoms onset compared to RT-PCR, ELISA was used as an alternative standard assay to evaluate LFIA performance. However, since LFIA is qualitative and ELISA results are quantitative, any IgM or IgG OD reading that exceeded the ELISA cut-off value was considered positive as a qualitative measure of antibodies. Figure 3 summarizes the IgM and IgG antibodies results detected by the seven LFIA assays included in our study compared to the in-house ELISA. While no false positive results were observed out of the 15 healthy subjects consistent with the ELISA, several of the RT-PCR confirmed cases that showed no antibody response by the in-house ELISA were found seropositive by many of the LFIA devices (Figure 3). As shown in Figure 4A, IgM antibodies sensitivity ranged from 0% to 100% at early points and from 32% to 91% at late time points after symptoms onset. IgG sensitivity of the seven LFIAs during the first week post symptoms onset ranged from 0% (95% CI 0-82%) to 66% (95% CI 30-94) and from 58% (95% CI 41-73%) to 93% (95% CI 68-99%) at late time points post symptoms onset (Figure 4B). The overall specificity for IgM and IgG detection by the seven LFIAs ranged from 70% (95% CI 52-83%) to 100% (95% CI 85-100%) as shown in Figure 4C.