Nanobody Selection against Complement C4b

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)).