Stop reagent for tmb substrate

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).

ELISA plates were coated with different concentrations of E. coli-RBD, Insect-RBD and HEK-293-RBD proteins. After washing and blockading of the free protein-binding sites with PBS—0.05% Tween20—and 3% BSA, different concentrations of rat serum (immunized with COVID-eVax vaccine) or anti-SARS-CoV-2 Spike S1 Subunit antibody (Sino Biological, 40150-T62) were added to each well and incubated overnight at 4 °C in PBS—0.05% Tween20—and 1% BSA. After washing, AP-conjugated goat anti-rat IgG antibody (SIGMA A8438) or AP-conjugated goat anti-rabbit IgG antibody (SIGMA A8025, Burlington, MA, USA) was added and the plates were further incubated for 1 h at RT. Finally, 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System (Sigma T8665) or alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for Elisa (Sigma P7998) was added as a substrate. After 30 min, the TMB reaction was stopped with the stop reagent for TMB substrate (Sigma S5814) and the absorbance was measured at 450 nm, while the pNPP reaction was measured at 405 nm at different time points. The optimal cut-off value was determined using the formula Cutoff = 2× + 3 S.D., where x is the mean and S.D. is the standard deviation of three independent negative-control readings. To discriminate positive results from background readings, the obtained highest cut-off value was chosen to be represented on the graphs.