Protein a beads

Heavy- and light-chain variable domain genes (S4 Table) were cloned into expression vectors and transfected into HEK-293F cells (ThermoFisher Scientific, R79007) using polyethylenimine (Polysciences, Inc. 23966) and cultured in serum-free expression medium (SinoBiological, M293TII) [59 (link)]. For production of dimeric IgA (dIgA) and pentamer IgM, the heavy and light chain plasmids were co-transfected with plasmid expressing the connecting J chain. Cell culture supernatant was collected 5 days after transfection and then assayed to determine neutralizing activity. IgG mAbs were purified by using protein A beads (Smart-Lifesciences, SA015025) and then assayed to determine antigen binding and neutralization. IgA and IgM mAbs that neutralize EV-A71 were purified, those with VkI, VkIII and VkIV were purified by using protein L resin (Genescript, L00239), the others were purified by mixed-mode chromatography and anion-exchange chromatography as described previously [51 (link)].

After transfection, the HEK293 cells were collected and rotated at 1000 rpm for 5 min to collect the supernatant only. The supernatant was mixed with protein A beads (Smart-Lifesciences) and slowly rotated at 4°C overnight. The mixture was put into an affinity column. The antibodies were washed down by eluent. The eluates were collected and concentration was determined by Nano-300.

The Technology Center for Protein Research at Tsinghua University provided the HEK293‐F cell line and the pVRC8400_tPA plasmid, while the pCDNA3.1 plasmid was purchased from Invitrogen (Thermo Fisher Scientific, USA). To express N1H (a model anti‐VEGF nanobody obtained from a Patent
²⁶ ), N2H‐9GS (a bivalent nanobody with two N1H nanobody connected by a GGGSGGGGS linker), we cloned them into the pVRC8400_tPA plasmid, while the pCDNA3.1 plasmid was used to clone the Fc‐fused multivalent nanobody (nanoFc). HEK293‐F cells were cultured in SMM 293‐TII medium (Sinobiological, China) at 5% CO₂ and 37°C for 4–5 days. The cells were transfected with the plasmid and PEI (Polysciences, USA) at a final concentration of 1 μg/mL and 3 μg/mL, respectively, at a density of 1.5–2.0 × 10⁶ cells/mL after being sub‐cultured for 3–4 passages. N1H and N2H‐9GS were purified using Ni beads and protein A beads (Smart Life sciences, China) were used to purify nanoFc in the supernatant. The purity of the protein was evaluated using SDS‐PAGE or size‐exclusion high‐performance liquid chromatography (SEC‐HPLC), while the concentration was measured by UV absorbance at 280 nm (Nanodrop 2000, Thermo Scientific, Wilmington, DE, USA). The concentration analyzer Lunatic (Unchained Labs, USA) was employed to determine the concentration of nanoFc when it was over 50 mg/mL.