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]).