Binding ELISA assay
To test if the vaccine can bind the relevant human receptor ACE2, the
plates were coated with 1µg/ml of ACE2 in PBS at a volume of 50µl/well.
The plate was incubated at 4oC overnight. The plate
was washed with PBS, Tween 0.01%. Added 50µl/well of Superblock
solution (Thermo Fisher, 37518) and incubated for 1h at RT on a shaker.
The blocking solution was flicked off and 50µl of the
CuMVTT-RBM or CuMVTT-VLPs at 1µg/ml were
added to the first row of the plate followed by 1:3 dilution. The plate
was incubated for 1h at RT, washed with PBS+Tween 0.01%. 50µl of mouse
anti-CuMVTT monoclonal antibody (clone 1-1A8/ batch 2)
at a concentration of 1µg/ml was added to each well as a secondary
antibody and incubated for 1h at RT on a shaker. The plate was washed
and 50µl of the detection antibody; HRP labelled goat anti-mouse IgG Fc
gamma at a dilution of 1:1000 in PBS-Casein 0.15% was added to each
well. The plate was incubated for 1h at RT. The plate was developed and
OD450 reading was performed (BioTek, USA).