SARS-CoV-2 Antibody Titration by ELISA

Individual sera were titrated in parallel for the presence of SARS‐CoV‐2 S1 RBD‐specific antibody by end‐point ELISA. The ELISA plates were functionalized by coating with recombinant Sars‐CoV‐2 S1 subunit protein (RayBiotech, #230‐30162) at a concentration of 2 μg/ml and incubated overnight (O/N) at 4°C. Subsequently, the plates were blocked with 3% fat‐free milk and 0.05% Tween20 in PBS for 1 h at RT. The sera were then added at a dilution of 1/20 (sera from day 7) or 1/500 (sera from day 14, 21, and 28) and diluted 1:10 up to 1/1,280 or 1/32,000, respectively, in duplicate, and the plates were incubated for 2 h at RT. After five washes with 0.05% Tween20 in PBS, the secondary anti‐murine IgG conjugated with horseradish peroxidase (HRP, PerkinElmer, #NEF822001EA) (1:2,000) was added and the plates were incubated for 1 h at RT. After washing, the binding of the secondary was detected by adding the substrate 3,3′,5,5′‐tetramethylbenzidine (TMB, BD Biosciences). The reaction was blocked with 0.5 M H₂SO₄ and the absorbance at 450 nm and reference 630 nm was measured.
Individual sera were titrated in parallel for the presence of VSV‐specific IgG by end‐point ELISA. Neutralizing dose 50 were measured as described (Sammicheli et al, 2016 (link)).