Nickel nta agarose

Human sera from heart transplant patients were obtained at Oregon Health & Science University with patient consent (OHSU IRB Protocol #0004474 as described previously [48 (link)]. These sera were pooled from 5 or 6 donors then diluted to NT₁₀₀ titers in Opti-MEM without FBS and incubated with 1 μg soluble trimer [48 (link)], or 0.5 mM of peptide for 1 h at 37°C. Either 15 μl of nickel NTA agarose (Invitrogen) or streptavidin agarose (EMD Millipore) was added to the samples followed by incubation at room temperature for 1 h with agitation. The sera were then centrifuged through affinity purification spin columns (Pierce) at 1000 × g for 30 sec to separate the sera from agarose. These sera were then tested in HCMV neutralization assays as described above.

For expression of the RBD subdomain of the SARS-CoV-1 (residues Arg306 to Phe527) and SARS-CoV-2 (residues Arg319 to Phe541) spike protein and human ACE2, genes encoding the proteins were synthesized and cloned upstream of either an Fc tag or rCD4-Avi tag, both with an additional 6 x His tag in mammalian expression vectors (2 (link)). The constructs were expressed in Expi293F™ cells (Thermo Fisher, A14527) and purified by affinity chromatography using Nickel-NTA agarose (Invitrogen, R90115). SARS-CoV-2 S1 protein with a His tag (S1N-C52H3) and SARS-CoV-2 S protein active trimer with His tag (SPN-C52H8) were separately purchased from ACRO biosystems. MERS-CoV S1 with a His tag was purchased from Sino Biological (40069-V08H).
All antigens with an Avi tag were biotinylated enzymatically using BirA biotin-protein ligase (Avidity, Bulk BirA) while non-Avi tagged antigens were biotinylated chemically using EZ-Link Sulfo-NHS-Biotin (Thermo Fisher, A39256). V_H and V_L sequences of SARS-CoV-2 control antibodies (COV2-2196, COV2-2130, S309, B38, H4, and CR3022) were obtained from the CoV-AbDab database (3 (link)) and cloned into pINT3/pINT54 IgG expression vectors (4 (link)) as synthetic gene fragments. The antibodies were expressed using the Expi293™ system and purified using Protein-A affinity chromatography (Generon, PC-A100). Control antibody SAD-S35 was purchased from ACRO biosystems.