PMC:7376845 / 24734-27108 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":"Utility of monoclonal antibody 1A9 for detection of S protein in a sandwich ELISA format and in SARS-CoV-2 infected cells\nBased on indirect ELISA data, mAb 1A9 has the strongest binding to S protein when compared with the other three mAbs. Hence, a sandwich ELISA was performed to determine if it can be paired with the human mAb CR3022 which is known to bind to the S1 subunit of SARS-CoV-2. As shown in Figure 4A, recombinant S protein was detected at 15.6 ng/mL and above when 1A9 was used as a capture antibody and CR3022 was used as a detector antibody. Since the S protein was His-tagged, a His-tagged haemagglutinin (HA) protein of influenza A virus was used to check for specificity of binding. The absorbance readings in the presence of S protein were significantly higher than that in the presence of HA for protein concentrations of 15.6 ng/mL and above.\nFigure 4 Performance of monoclonal antibody 1A9 for detection of (A) S protein in a sandwich ELISA format and (B) SARS-CoV-2 infected cells\nHA: haemagglutinin; H7N7: influenza A (H7N7); mAb: monoclonal antibody; MOI: multiplicity of infection; OD: optical density; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.\nA. Sandwich ELISA assay to determine mAb 1A9 ability to pair with the human mAb CR3022 for the detection of a His-tagged SARS-CoV-2 spike protein. 1A9 and CR3022 were used as capture and detector antibodies respectively. His-tagged HA protein of influenza A (H7N7) virus was used as a negative control. Averaged readings across three replicate experiments are presented. Error bars represent standard deviations across the three replicate experiments. Asterisks indicate significantly increased binding of the antibody pairs to SARS-CoV-2 S protein compared to influenza A (H7N7) HA at p \u003c 0.05.\nB. Vero E6 cells were mock-infected (left panel) or infected with SARS-CoV-2 (right panel; MOI of 1). At 24 hour post infection, the cells were stained with mAb 1A9 (5 µg/mL) followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Next, 1A9 was tested on SARS-CoV-2-infected Vero-E6 cells. As shown in Figure 4B, mAb 1A9 stained a considerable number of SARS-CoV-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of S protein during 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