PMC:7376845 / 19302-21937 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7376845","sourcedb":"PMC","sourceid":"7376845","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7376845","text":"Four murine monoclonal antibodies bind to a fragment of the spike protein of SARS-CoV-2\nFour mAbs with distinct binding profiles to SARS-CoV-1, as previously mapped by internal deletion mutagenesis study, were selected for testing to determine if they cross-react with SARS-CoV-2. A fragment containing residues 1048 to 1206 of SARS-CoV-2 S protein was expressed in 293FT cells via transient transfection and WB analysis was performed using the four mAbs, namely 2B2, 1A9, 4B12 and 1G10. As shown in Figure 2A, all four mAbs detected this fragment of SARS-CoV-2, which is consistent with the sequence alignment shown in Figure 1. Due to the easy detachment of 293FT cells, COS-7 cells were used for IF assay instead. IF analysis performed on transiently transfected COS-7 cells showed binding of the four mAbs to this S protein fragment of SARS-CoV-2 (Figure 2B). These interactions are also specific for the SARS-COV-2 S protein (1048–1206) fragment as all four mAbs did not show binding to the negative control HBcAg.\nFigure 2 Monoclonal antibodies expected to target a SARS-CoV-2 S protein S2 fragment, (A) hybridise to the peptide fragment in western blot and (B) recognise cells expressing the peptide as shown by immunofluorescence\naa: amino acid; HBcAg: hepatitis B virus core antigen; DAPI: 4′,6-diamidino-2-phenylindole; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; S protein: spike protein; vec: pXJ40-Myc expression vector used as an empty vector control.\nA. Each photo depicts a western blot using the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). Empty vector-transfected 293FT cell products are on the lanes labelled as ‘Vec’, while products of 293FT cells expressing Myc-tagged SARS-CoV-2 S protein fragment (aa 1048–1206; SARS-CoV-2 numbering) or Myc-tagged HBcAg are on the lanes respectively labelled ‘S’ or ‘HBcAg’. Primary antibodies were labelled with horseradish peroxidase-conjugated secondary antibodies. A ladder indicative of the molecular weights in kD of the proteins relative to their vertical position on the blots, is indicated on the left of the panel.\nB. Each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (Myc, 2B2, 1A9, 4B12, or 1G10). Immunofluorescence assay results of COS7 cells expressing Myc-tagged HBcAg (top photos) or a Myc-tagged fragment (aa1048–1206) of SARS-CoV-2 S protein (bottom photos) using the indicated primary antibodies, followed by Alexa Fluor 488-conjugated secondary antibody (green) are shown. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 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