ELISA Protocol for Anti-RBD Antibody Detection

Anti-RBD antibodies in sera were detected by enzyme-linked immunosorbent assay (ELISA) as described previously (34 (link), 40 (link), 44 (link)). Briefly, high binding 96-well Maxisorp plates (Thermo Scientific) were coated overnight at 4°C with 1 µg/mL of an RBD target in PBS. Plates were rinsed three times with PBS and 0.05% Tween-20 (J62844, Alfa Aesar) then blocked for 1 hour at room temperature with 1% bovine serum albumin (BSA) in PBS (Thermo Scientific 37525). Plates were rinsed three times before addition of diluted sera or nasal washes and incubated overnight at 4°C. After rinsing three times as above, anti-mouse IgG (0107-05, SouthernBiotech), anti-mouse IgG1 (1071-05, SouthernBiotech), anti-mouse IgG2a (1081-05, SouthernBiotech), or anti-mouse IgA (1165-05, SouthernBiotech) conjugated with horseradish peroxidase (HRP) were added to plates and incubated for 1 hour at room temperature. Colorimetric signals were developed with 3, 3′, 5, 5′ - tetramethylbenzidine (TMB) (T0440, Sigma) and reactions were stopped with 2N HCl. Antibody levels were quantified via absorbance readings at 450 nm and 570 nm on a microplate reader (Cytation 5; Biotek, Winooski, VT).