Fig. 3

Validation of COVID-19 disease-specific B-cell epitopes derived from biopanning of phage-displayed random peptide libraries. a Serological detection of COVID-19 convalescent patients using NP-SP11 and NP-SP11 mutant peptides. Peptide (2.5 μg/well; 10 μg/ml) was coated overnight followed by serum incubation. Patient IgG binding to peptide was detected by anti-human IgG HRP secondary antibody, and the OD was measured at 450 nm. Sequence of AS3-3-42 phage-displayed peptide and synthetic SARS-CoV-2 NP protein peptides with TLPK motif (NP-SP11) or the GAGA-mutated motif (NP-SP11 mutant) are shown. b Peptide competition assay. IgG from AS3 patient serum was captured, followed by 1-h incubation with both 1 × 10⁹ pfu AS3-3-42 phage (or control phage) and different amounts of synthetic NP peptide. Phage binding was detected by anti-M13 HRP secondary antibody and OD was measured at 450 nm. c Electrostatic surface of the SARS-CoV-2 NP N-terminal domain (NP-NTD, PDB ID: 6M3M). The potential charge distribution was calculated by PyMOL. Blue color indicates positive charge potential; red color indicates negative charge potential. Dashed line denotes the potential ribonucleotide binding pocket [14]. TLPK residues are shown as a sphere. Horizontal dashed line denotes the cutoff value (calculated as mean OD₄₅₀ + 4 × SD of normal healthy serum samples) for AS3-3-42 phage or NP-SP11 peptide. * Indicates COVID-19-specific positive case