Development of a Fast SARS-CoV-2 IgG ELISA, Based on Receptor-Binding Domain, and Its Comparative Evaluation Using Temporally Segregated Samples From RT-PCR Positive Individuals

Abstract

SARS-CoV-2 antibody detection assays are crucial for gathering seroepidemiological information and monitoring the sustainability of antibody response against the virus. The SARS-CoV-2 Spike protein’s receptor-binding domain (RBD) is a very specific target for anti-SARS-CoV-2 antibodies detection. Moreover, many neutralizing antibodies are mapped to this domain, linking antibody response to RBD with neutralizing potential. Detection of IgG antibodies, rather than IgM or total antibodies, against RBD is likely to play a larger role in understanding antibody-mediated protection and vaccine response. Here we describe a rapid and stable RBD-based IgG ELISA test obtained through extensive optimization of the assay components and conditions. The test showed a specificity of 99.79% (95% CI: 98.82–99.99%) in a panel of pre-pandemic samples (n = 470) from different groups, i.e., pregnancy, fever, HCV, HBV, and autoantibodies positive. Test sensitivity was evaluated using sera from SARS-CoV-2 RT-PCR positive individuals (n = 312) and found to be 53.33% (95% CI: 37.87–68.34%), 80.47% (95% CI: 72.53–86.94%), and 88.24% (95% CI: 82.05–92.88%) in panel 1 (days 0–13), panel 2 (days 14–20) and panel 3 (days 21–27), respectively. Higher sensitivity was achieved in symptomatic individuals and reached 92.14% (95% CI: 86.38–96.01%) for panel 3. Our test, with a shorter runtime, showed higher sensitivity than parallelly tested commercial ELISAs for SARS-CoV-2-IgG, i.e., Euroimmun and Zydus, even when equivocal results in the commercial ELISAs were considered positive. None of the tests, which are using different antigens, could detect anti-SARS-CoV-2 IgGs in 10.5% RT-PCR positive individuals by the fourth week, suggesting the lack of IgG response.

Longitudinal Serology of SARS-CoV-2-Infected Individuals in India: A Prospective Cohort Study

“…Anti-SARS-CoV-2 RBD IgG antibody was detected using an ELISA as described previously. 5 Briefly, samples and controls were diluted to 1:50 in the sample diluent, and 100 μL was added per well of the stabilized RBD-coated plate. After 30 minutes of incubation at room temperature (23 ± 2°C), the plates were washed six times with wash buffer.…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…Some studies showed that S protein-based ELISA test was less sensitive to detect IgG and IgA against SARS-CoV-2 (<70%) [206] , [207] while few other studies reported that such test was highly sensitive (at least 90%) to detect IgG, IgA, or IgM against the virus S antigen [199] , [204] , [205] , [208] . Therefore, N protein-based ELISA assay could be a better choice of ELISA test to predict serological response in the SARS-CoV-2 patients as the test results of this assay seem to be more consistent and vary less in different reported studies [202] , [203] as compared to the S protein-based ELISA test [199] , [204] , [206] , [207] , [208] , [209] . On the other hand, some studies had reported the use of ELISA test that was aimed to detect different antibodies against different proteins of SARS-CoV-2, and examples of these tests were tests that were focusing to detect antibodies against N and S protein of SARS-CoV-2 [196] , [204] , [209] , [210] , [211] , [212] , [213] , [214] , [215] .…”

“…Therefore, N protein-based ELISA assay could be a better choice of ELISA test to predict serological response in the SARS-CoV-2 patients as the test results of this assay seem to be more consistent and vary less in different reported studies [202] , [203] as compared to the S protein-based ELISA test [199] , [204] , [206] , [207] , [208] , [209] . On the other hand, some studies had reported the use of ELISA test that was aimed to detect different antibodies against different proteins of SARS-CoV-2, and examples of these tests were tests that were focusing to detect antibodies against N and S protein of SARS-CoV-2 [196] , [204] , [209] , [210] , [211] , [212] , [213] , [214] , [215] . Compared to molecular detection using the qRT-PCR method that showed more consistent test sensitivity and specificity across different reported studies as an example [71] , [72] , [87] , [88] , the sensitivity and specificity of ELISA assays in detecting various antibodies (IgG, IgA, and IgM) against different antigen proteins of SARS-CoV-2 demonstrated a high degree of variations in which some studies reported low test sensitivity (<60%) [183] , [196] , [216] and specificity (as low as below 80%) [215] whereas some studies would report high test sensitivity and specificity (>85%) [210] , [211] .…”

“…Compared to molecular detection using the qRT-PCR method that showed more consistent test sensitivity and specificity across different reported studies as an example [71] , [72] , [87] , [88] , the sensitivity and specificity of ELISA assays in detecting various antibodies (IgG, IgA, and IgM) against different antigen proteins of SARS-CoV-2 demonstrated a high degree of variations in which some studies reported low test sensitivity (<60%) [183] , [196] , [216] and specificity (as low as below 80%) [215] whereas some studies would report high test sensitivity and specificity (>85%) [210] , [211] . Even though the ELISA test might be less sensitive and specific in detecting the presence of SARS-CoV-2 based on the findings from few studies [196] , [209] , [215] , [217] , however, this assay was reported to have higher sensitivity and specificity than other serological tests like IFA in detecting the presence of SARS-CoV-2 [183] , [184] .…”

Sequential Profiling of Anti-SARS CoV-2 IgG Antibody in Post COVID-19 Patients

“…The uniqueness of RBD region among the coronavirus families, makes the anti-RBD IgG specific to each species. Hence the antibodies against RBD possess more neutralizing effect on the virus and are considered to be the most suitable antibody for the assessment of immunity against COVID-19 [6][7][8][9][10][11].…”