Pierce streptavidin coated high capacity plates

To identify the enhancing epitope of the mAbG3 on the PEDV S1 subunit, peptide binding ELISA was performed. Five biotin-labeled 12 amino acid peptides of the PEDV S1 subunit that matched with the mAbG3-bound phage mimotopes and a control peptide were synthesized (Genscript, Piscataway, NJ, United States). Individual peptides were diluted in PBS to 10 μg/ml, and 100 μl of the diluted peptides were added to the separate wells (triplicate) of the streptavidin-coated microplate (Pierce^™ Streptavidin Coated HighCapacity Plates; Thermo Fisher Scientific), and the plate was kept at 4°C overnight. The plate was washed three times with TBST, and the empty sites of the well surface were blocked with 5% skim milk in TBST. After 1 h at 37°C, the fluids were discarded. The wells were washed again, and 1 μg of purified mAbG3 in 100 μl of TBST was added to the individual wells. Buffer was included as a negative antibody control. The plate was incubated at 37°C for 1 h. After washing, 100 μl of HRP-labeled mouse IgG-kappa binding protein (Santa Cruz Biotechnology, Dallas, TX, United States; 1:1,000 in TBST) was added to each well, and the plate was incubated further at 37°C for 1 h. After washing, ABTS substrate (KPL; 100 μl) was added to each well, and the plate was kept in darkness at room temperature for 30 min. The OD of individual wells was measured at 405 nm (BioTek spectrometer).

For C4b nanobody selection, a nanobody library was panned on purified C4b in PBS and immobilized on streptavidin-coated plates. To this end, C4b at a concentration of 5.1 µM was incubated with a 40× molar excess of EZ link Maleimide-PEG2-Biotin (Thermo Fisher Scientific, 210901BID) in PBS, pH 7.4, overnight at 4°C. Unreacted linker was separated with a Bio-Spin 6 column (Bio-Rad Laboratories) that was equilibrated in PBS. Plates were coated with 1 µg of biotinylated C4b per streptavidin‐coated well (Pierce Streptavidin‐Coated High‐Capacity Plates, 15501; Thermo Fisher Scientific) after incubation at 4°C for 16 h in PBS. The immobilized target was panned with 7 × 10⁹ phages preblocked with 2% (w/v) milk for 2 h at room temperature (RT), followed by several 0.05% (v/v) Tween 20/PBS and PBS washes. Remaining bound phages were eluted by incubation of the wells with 0.1 M triethylamine, pH 12.0, for 30 min at RT while shaking. Eluates were then neutralized by addition of 1 M Tris-HCl, pH 7.4, before reinfection of TG1 bacteria. The resulting enriched phage library was submitted to a second round of selection. From the second selection round output, 94 single colonies of TG1 bacteria were picked and screened on streptavidin-immobilized C4b plates following a previously described phage ELISA protocol (47 (link)).