Antibody Validation for SARS-CoV-2 ORF8
Category: Research Poster
Author(s): Ashlee Burns, Brandon Schout, Katherine Flores
Presenter(s): Ashlee Burns, Brandon Schout, Katherine Flores
Mentors(s): Alan Schenkel, William Schountz
The Open Reading Frame 8 (ORF8) gene is highly conserved across many strains of the SARS-CoV-2 virus. The ORF8 protein is hypothesized to be involved in suppression of the antiviral immune response. ORF8 prevents MHC I from reaching the cell surface and blocks interferon response factor signaling via IRF3 binding. Verification of the presence of the ORF8 protein in infected cells is still in progress using Western Blot assays and immunofluorescence. Repeated attempts failed to produce the desired results in blots so we began troubleshooting the reagents. Blotting reagents directly onto a nitrocellulose membrane showed issues with secondary antibodies, but did not provide any answer about issues with the ORF8 recognition by the primary antibodies There was a potential signal indicating that the reagents interacted with a synthetic ORF8 protein, although the signal was weak. A more thorough test was then performed with the 96-well plate, which did not confirm the presence of the ORF8 protein, but did work as expected with positive controls. Once troubleshooting is completed and the presence of the protein is able to be verified, we will begin analyzing cell expression of the ORF8 protein, as well as MHC Class 1 and IRF3 in human and bat cell.