FIG 2.

Interactions between coronavirus spike and RBD-specific MAb. (A) ELISA for detection of the binding between MERS-CoV RBD-specific MAb (i.e., Mersmab1) and MERS-CoV spike ectodomain (S-e). Mersmab1 was precoated on the plate, and recombinant S-e or RBD was added subsequently for ELISA. Binding affinities were characterized as ELISA signal at an optical density (OD) at 450 nm. PBS was used as a negative control. (B) ELISA for detection of the binding between Fab of Mersmab1 and MERS-CoV S-e. Recombinant S-e was used to precoat the plate, and Mersmab1 or Fab was added subsequently for ELISA. (C) Flow cytometry for detection of the binding between MERS-CoV S-e and DPP4 receptor and among S-e, Mersmab1, and CD32A (i.e., Fc receptor). Cells expressing DPP4 or CD32A were incubated with S-e alone, S-e plus Mersmab1, or S-e plus a SARS-CoV RBD-specific MAb (i.e., 33G4). Fluorescence-labeled anti-His₆ antibody was added to target the C-terminal His₆ tag on S-e. Cells were analyzed using fluorescence-activated cell sorting (FACS). (D) The expression levels of cell-membrane-associated DPP4 and CD32A were characterized using Western blotting targeting their C-terminal C9 tag and then used to normalize the binding affinity as measured in panel C. As an internal control, the expression level of cellular actin was measured using an anti-actin antibody. All of the experiments were repeated at least three times, with similar results, and representative results are shown. Error bars indicate SD (n = 5). Statistical analyses were performed as a one-tailed t test. ***, P < 0.001. Mersmab1 and its Fab both bind to MERS-CoV RBD and S-e.