Linear epitope mapping with peptide-ELISA assay

The 18-mer C-terminal biotinylated synthetic peptides, spanning the full length of SARS-CoV-2 N protein, were synthesized by Sangon Biotech (Shanghai). The reactivity of synthetic peptides with mAb nCoV396, nCoV454, nCoV457, and nCoV416 were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 100 μL/well of individual peptide (10 μg/mL in Phosphate-Buffered Saline (PBS) buffer) at 4 °C overnight. An unrelated mAb TT017 were used as a negative controls and streptavidin as a positive control. The wells were then incubated sequentially with 100 μL/well of 1x phosphate-buffered saline, 0.1% Tween 20 Detergent (PBST) plus 5% skimmed milk powder at 37 °C for 1 h, and 1 μg of mAb at 10 μg/mL was incubated at 37 °C for 1 h. Goat anti-human IgG-horseradish peroxidase (HRP, 1:10,000 dilution; Promega, W4031) in PBST plus 1% milk powder was used as secondary antibody at 37 °C for 1 h. Five washes with PBST were carried out between incubation steps. For color development, 100 μL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) mixture was added and incubated for 10 min, followed by addition of 50 μL/well of 1 M H₃PO₄ to stop the reaction. Absorbance was measured at 450 nm in a 96-well plate reader.