SARS-CoV-2 Antibody Detection by ELISA

Patient plasma samples were tested for the presence of anti-SARS-CoV-2 reactive IgM or IgG antibodies as described previously (Snyman et al., 2021 (link)). ELISA plates were coated with 500 ng/ml of the D614G ancestral virus receptor binding domain (RBD) (GenBank: MN975262; provided by Dr Galit Alter, Ragon Institute, Cambridge, MA, USA) overnight at 4°C. Then blocked with 1% BSA-TBS at room temperature (RT) for 1 hr, followed by samples diluted at 1:100 in BSA-TBS+0.05% Tween 20 for 1 hr at RT. Secondary anti-IgM or -IgG antibodies (Jackson ImmunoReasearch, West Grove, PA, USA) were added at 1:5000 diluted in BSA-TBS+0.05% Tween 20 and incubated again for 1 hr at RT. Finally, plates were developed with one-step Ultra TMB substrate (Thermo Fisher Scientific) for 3 or 5 min respectively and signal development was stopped with the addition of 1 N H₂SO₄. Plates were washed with TBS+0.05% Tween 20 between each incubation step. All signals were compared to anti-SARS-CoV-2-specific monoclonal IgG (clone CR3022) or IgM (clone hIgM2001). Pre-pandemic plasma samples were used as negative controls to determine seroconversion cut-offs calculated as three times the standard deviation plus the mean.