Recombinant sars cov 2 s1 subunit protein

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⁻¹ and incubated overnight (O/N) at 4 °C. Subsequently, the plates were blocked with 3% fat-free milk, 0.05% Tween 20 in PBS for 1 h at room temperature. The sera were then added at a dilution of 1/20 (sera from day 7) or 1/500 (sera from days 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 room temperature. After five washes with 0.05% Tween 20 in PBS, the secondary anti-mouse IgG conjugated with horseradish peroxidase (PerkinElmer, NEF822001EA; 1:2,000) was added and the plates were incubated for 1 h at room temperature. After washing, the binding of the secondary antibody was detected by adding the substrate 3,3′,5,5′-tetramethylbenzidine (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 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)).

We have obtained the Recombinant SARS-CoV-2, S1 Subunit Protein (RBD), and Recombinant SARS-CoV-2 Nucleocapsid Protein from Raybiotech (Peachtree Corners GA, cat# 230-30162-1000 and 230-30164-500 correspondingly). The proteins were labeled using Alexa Fluor™ 546 Protein Labeling Kit from Thermo Scientific (cat# A10237) following manufacturer directions except for the RBD that was purified from the dye with Amicon-Ultra 10K cutoff filters (Merck, Millipore cat#UFC201024) instead of the column provided by the kit has a restrictive MW of 50KD (the recombinant RBD protein was 25KDa). The recovery of the protein and labeling efficiency was measured using nanodrop and was about 80% recovery and 0.02 dye molecules per aminoacid. We added 0.5 μg per well of protein.