Anti mers cov spike protein antibody

RoNi/7.1 or Huh-7 cells were incubated with different concentrations (0, 1.25, 2.5, 5, 10, 20 µg/ml) of goat anti-human CD26/DPP4 antibody (R&D Systems, Minneapolis, MN) or control goat IgG antibody at 37°C for 1 h. Antibody-treated cells were exposed to MERS-CoV/EMC at an MOI of 1 at 37°C for 1 h in the presence of antibodies. Virus-antibody inocula were then removed, cells were washed in 0% DMEM, fresh DMEM (2% FBS) was added, supernatants were harvested 24 h post-exposure, and viral yields were determined by plaque assay. At the same time, plates were fixed with 10% NBF and then stained with rabbit polyclonal anti-MERS-CoV spike protein antibody (Sino Biological, Beijing, China) followed by secondary Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Life Technologies, Carlsbad, CA). Hoechst 33342 dye was used to stain nuclei. The percentage of infected cells was measured and analyzed using the Operetta high content imaging system (PerkinElmer Waltham, MA) and analysis software (Harmony 3.1).

Vero cells (1.2 × 10⁴ cells) were seeded in black 384-well culture plates (Corning Inc., Corning, NY, USA) containing DMEM, supplemented with 1× antibiotic–antimycotic solution (100 units/mL of penicillin, 100 μg/mL of streptomycin, and 250 ng/mL of amphotericin B, Gibco) and 2% FBS. After 24 h, the indicated concentration of compounds and SARS-CoV-2 (0.0125 multiplicity of infection, MOI), SARS-CoV (0.05 MOI), or MERS-CoV (0.0625 MOI) was added. After 24 h of incubation, the cells were fixed in 4% paraformaldehyde, and immuno-stained with anti-SARS-CoV-2 nucleocapsid protein antibody, anti-SARS-CoV spike protein antibody, or anti-MERS-CoV spike protein antibody (Sino Biological Inc., Beijing, China). Then, the cells were incubated with anti-rabbit IgG secondary antibody and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). The stained images were acquired using an Operetta^® High-Content Imaging System, and analyzed using Image-Mining 3.0 plug-in software (20×; PerkinElmer, Inc., Waltham, MA, USA), as previously described [30 (link)].