Recombinant Antibody Generation and Purification

Recombinant antibodies were generated using the Expi293 (ThermoFisher) or Expi293 FUT8^−/− system using previously described protocols (47 (link)). Briefly, an equal ratio of heavy- and light-chain plasmids was complexed with ExpiFectamine in OptiMEM and added to Expi293 cells in culture at 3 × 10⁶ cells/ml. Enhancer 1 and Enhancer 2 were added 20 h after transfection. After 6 d, recombinant IgG antibodies were purified from cell-free supernatants by affinity purification using 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 were more than 95% pure and endotoxin levels were less than 0.05 EU mg⁻¹, as measured by the Limulus amebocyte lysate assay. Purified IgG was fluorescently labeled with Alexa Fluor 647-NHS or FITC-NHS (ThermoFisher) at a 15-fold molar excess for 1 h at room temperature and double-dialyzed into PBS.