Indirect Immunofluorescence Assay for MERS-CoV Detection

For analysis of the indirect immunofluorescence assay, a mixture of MERS-CoV-infected and non-infected Vero cells at a ratio of 3:1 was seeded onto slide glasses. The cells were then fixed with acetone and incubated with the normal mouse IgG, 492-1G10E4E2 or 506-2G10G5 monoclonal antibody at 37°C for 2 h. The samples were further incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific Co.). Finally, the samples were mounted and analyzed using a fluorescence microscope (1×70, Olympus Co.) (28 (link), 29 (link)). To visualize the confocal microcopy, Vero cells (5 × 10⁴) were seeded onto cover glasses in 12 well plates and infected with MERS-CoV (0.1 MOI). After two days, the infected cells were fixed with 4% paraformaldehyde and subsequently blocked with 1% BSA and 0.1% triton X-100 in PBS. The slides were incubated in the presence of the 492-1G10E4E2 or 506-2G10G5 monoclonal antibody for 2 h, washed and then incubated with the Alexa Flour 488-conjugated goat anti-mouse IgG antibody for 1 h. Hoechst 33258 (Thermo Fisher Scientific) was used to stain the nuclei. The slides were examined by Carl Zeiss LSM710 (Carl Zeiss Co. Ltd.).