ELISA Assay
Corning half area 96-well plates were coated with 1 µg/ml
RBDWT or mutated RBDs in PBS overnight at 4°C and then
blocked with PBS/0.15% casein. Convalescent human sera were added,
serially diluted 1:3 and incubated on plates for one hour at room
temperature. Bound IgG antibodies were detected with goat anti-human
IgG-POX antibody (Nordic MUbio, Susteren, The Netherlands). ELISA was
developed with tetramethylbenzidine (TMB), stopped by adding equal 1 M
H2SO4 solution, and read at
OD450nm.