Panning and screening of the phage-display libraries

Three rounds of panning were performed using the target antigens, including recombinant S1-hFc and RBD-mFc. Briefly, phage-display libraries were incubated in 10 μg/mL target antigen-coated immunotubes for 1 h at room temperature (RT). After washing extensively with PBST, the bound phages were eluted with 0.1 M Gly-HCl buffer (pH 2.2). The eluted phages were used to infect E. coli for subsequent amplification and subjected to further selection rounds. In the second and third rounds of panning, the input phages or output phages were allowed to incubate once or twice with human or mouse IgG mixtures-adsorbed immunotube for 1 h at RT for Fc tag antibody depletion.

After panning, the output clones were picked, induced, and screened via monoclonal soluble scFv ELISA with the S1 protein and human IgG mixture, or the RBD protein and mouse IgG mixture, respectively. In brief, 96-well plates were coated with 2 μg/mL of S1 protein (100 μL per well), or 1 μg/mL of RBD protein (100 μL per well), and 1.5 μg/mL (100 μL per well) of human IgG mixture and mouse IgG mixture diluted in NaHCO₃ (pH 9.2) overnight at 4 °C. The coated plates were blocked with 2% milk for 1 h at RT and washed with PBST. Then, plates were incubated for 1 h with soluble scFv induced in the supernatant from individual clones. After incubation, plates were washed with PBST and incubated with HRP-conjugated anti-c-Myc antibody for 1 h at RT. Before reading, the plates were washed with PBST and developed with TMB peroxidase substrate solution. The reactions were stopped with 2 M HCl, and plates were read at 450 nm.