2.5 SARS-CoV-2 spike protein 1 and ACE2 binding inhibition by sera
and BALF of immunized mice
Two weeks after the last immunization, we analyzed inhibition ability of
Spike:ACE2 binding in serum and BALF by using Spike S1 (SARS-CoV-2):
ACE2 Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San
Diego, CA) according to the instructions provided by the manufacturer.
Briefly, the serum and BALF samples were first added to the 96-well
plate (Nunc) coated with Spike S1 then diluted 2-fold serially at 1/100
to 1/3200 and 1/2 to 1/64, respectively. Subsequently, ACE2-Biotin,
Streptavidin-HRP and TMB were reacted in the 96-well plate,
respectively. The chromogen produced was measured at an absorbance of
450 nm using a SpectraMax ABS PLUS. Binding inhibition (%) was obtained
from the following formula: Binding inhibition (%) = 100 × (([OD
value in the absence of specimen] - [OD value in the presence of
specimen]) ÷ [OD value in the absence of specimen]).