Liquid substrate system for elisa

Purified mAbs were conjugated to horseradish peroxidase using the Lightning-Link kit (Innova Biosciences) according to the manufacturer’s instructions. Microtitre plates were coated with purified mAbs, either individually or as a mixture, at 10 µg ml⁻¹ in coating buffer and stored at 4 °C overnight. After rinsing twice with PBS the plates were blocked with 4 % skimmed milk in PBS for 30 min. Overnight cultures were added to wells either directly or after dilution with PBS and incubated for 1 h. After five washes detection was by horseradish peroxidase-conjugated mAbs, again either separately or as a mixture, followed by substrate TMB (liquid substrate system for ELISA, Sigma). After 15 min results were recorded by scanning.

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.