Hrp conjugated anti human igg antibody

Enzyme-linked immunosorbent assay (ELISA) was used to test antibody binding to SARS-CoV-2 receptor binding domain (RBD) (catalog numbers 40592-V08H, Sino Biological US Inc., Wayne, PA, USA) (31 (link), 32 (link)). High-binding 96-half-well microplates (Corning Life Sciences, Tewksbury, MA, USA) were coated with 50ng RBD protein in PBS overnight at 4°C. The next day, plates were washed five times with PBS + 0.5% Tween 20 and treated with blocking buffer (PBS + 0.5% Tween 20 + 2% BSA + 2% normal goat serum [Jackson ImmunoResearch Inc., West Grove, PA, USA]) for two hours. Different dilutions of human plasma in blocking buffer were added and incubated for one hour. Plates were washed and incubated for one hour with HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch Inc.) (1:10,000 dilution). The process was stopped by adding 50µL of 1N sulfuric acid to 100L TMB substrate (Thermo Fisher Scientific, Rockford, IL, USA). BioTek Synergy 2 plate reader was used to measure OD at 450 nm (BioTek Instruments Inc., Winooski, VT, USA).

SDS-PAGE and immunoblotting were performed in accordance with our previous methods^{21 (link),24 (link)}. Briefly, samples were prepared with Laemmli buffer containing 5% 2-mercaptoethanol. SDS-PAGE was performed using Tris–glycine–SDS buffer (pH 8.3) and proteins were detected by Coomassie Brilliant Blue staining. For western blotting, proteins were transferred onto a PVDF membrane. After blocking nonspecific proteins, scR2agoTNF-Fc protein was probed with HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Visualization was performed by chemiluminescence with ECL Prime (GE Healthcare).