SARS-CoV-2 spike-specific antibodies were measured by ELISA as described previously [19 (link),20 (link),24 (link)]. Briefly, flat bottom 96-well ELISA plates (Nunc MaxiSorp Plates, Thermo Fisher Scientific, Karlsruhe Germany) were coated with 50 ng/well of one of the SARS-CoV-2 spike antigens listed in Supplementary Table S1 (from ACROBiosystems, Newark DE, USA and The Native Antigen Company, Kidlington, UK) overnight at 4 °C. Mouse sera were three-fold serially diluted in PBS containing 1% BSA (Carl Roth GmbH, Karlsruhe, Germany) (PBS/BSA), transferred to the coated ELISA plates and incubated for 1 h at 37 °C. Plates were then probed with goat anti-mouse IgG HRP diluted in PBS/BSA. SARS-CoV-2 spike-specific antibodies were detected using 3′3′,5′5′- Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, Taufkirchen, Germany), and the reaction was stopped using Stop Reagent for TMB Substrate (Sigma-Aldrich). The absorbance was measured at 450 nm with a 620 nm reference wavelength using the Spark® multimodal microplate reader (Tecan, Männedorf, Switzerland). Data were normalized using a positive control sample. The cut-off value for positive mouse serum samples was determined by calculating mean OD 450 nm values of the PBS control group sera plus six standard deviations (mean + 6 SD).
Free full text: Click here
