Recombinant Antibody Generation

Recombinant antibodies were generated using the Expi293 or Expi293 FUT8^−/− system (ThermoFisher) using previously described protocols^{27 (link)}. An equal ratio of heavy and light chain plasmids (or only nanobody-Fc) was complexed with Expifectamine in OptiMEM and transfected into Expi293 cells in culture at 3 × 10⁶ cells/ml. Enhancer 1 and 2 were added 20 h after transfection. Recombinant IgG was harvested and purified after 6 days with protein G sepharose beads (GE Healthcare), dialyzed in PBS, filter-sterilized (0.22 μm), concentrated with 100 kDa MWCO spin concentrator (Millipore), purified with Superdex 200 Increase 10/300 GL (GE Healthcare), and finally assessed by SDS–PAGE followed by SafeBlue staining (ThermoFisher). All antibody preparations had endotoxin levels were <0.05 EU mg⁻¹, as measured by the limulus amebocyte lysate assay.