2.7 Indirect ELISA
The 96-well microtiter plates (Corning Costar, USA) were coated with
recombinant truncated EBOV GP (1 μg/well) at 4 °C overnight, then
blocked with 1% BSA at 37 °C for 2 h. 100 μL serially diluted serum
samples were added into the wells, and incubated at 37 ℃ for 90 min.
After five washes, HRP-conjugated goat anti-horse IgG (Bios, China) was
added and incubated for an additional hour at 37 °C. Afterwards, 100 μL
of the substrate 3,3’,3,5’-tetramethyl benzidine (TMB) (Sigma, USA) was
added and incubated for 10 min at room temperature. After five washes,
and 50 μL of 0.5 M H2SO4 was added to
stop the reaction. The optical density (OD) values were measured at a
wavelength of 450 nm. Positive result was recognized as over 2.1 after
normalized by OD value to negative control.