Citation
- Authors: Mishra N. et al.
- Year: 2022
- Journal: J Mol Biol 434 167583
- Applications: in vitro / DNA / FectoPRO
- Cell type: HEK-293F
Method
Expression and purification of IgG proteins: Phage clone variable domain DNA was amplified by PCR and subcloned into pSCSTa-hIg1 and pSCST1-hk vectors. Vectors for the heavy and light chains were transfected into HEK293F cells using FectoPro according to the manufacturer’s instructions (Polyplus Transfection, NY). Cell cultures were incubated at 37 °C for 4-5 days post-transfection. The cell cultures were centrifuged, and the supernatants were applied to a protein-A affinity column (∼2 mL packed beads per 600 mL culture) (Pierce, ThermoScientific, Rockford, IL). IgG proteins were eluted with 100 mM glycine, pH 2.0 and neutralized with 2 M Tris, pH 7.5. The eluent underwent buffer exchange on PBS, pH 7.4, and concentrated by centrifugation in a 50 kDa centrifugal concentrator.
Abstract
The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.