M293tii

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)].

The VH and VL sequences of the IgG antibodies were amplified by RT-PCR and cloned into the vector plasmids AbVec2.0-hIgG1 (80795, Adgene, USA) for expressing the heavy chain, MapAbVec1.1-IGKC (80796, Adgene) for expressing the kappa light chain, and AbVec1.1-IGLC2 (99575, Adgene) for expressing the lambda light chain. Antibody expression plasmids (0.5 μg/ml) were co-transfected into 293F cells (1×10⁶ cells/ml) in SMM 293-TII expression medium (M293TII, SinoBiological) using PEI transfection reagent (23996-2, Polysciences, Pennsylvania, USA) and cultured for 6-7 days, and the cells and cell debris were removed by centrifugation at 4500 ×g and filtration (0.22 mm). The supernatant containing recombinant antibodies were purified using an ÄKTA express FPLC device using Protein A columns, washed with 20 ml of PBS, and eluted with glycine elution buffer (pH=2.0) into a collection tube containing Tris HCl (pH = 8.0). The purified antibody was dialyzed three times in PBS and stored at -40°C for later use. The purity of the antibodies were checked by running a polyacrylamide gel electrophoresis (PAGE) and visualized with Coomassie staining.

OverExpress^TME. coli C43(DE3) obtained from Weidi Biotechnology (Shanghai) was used to express the recombinant proteins.
Expi293F cells, obtained from Thermo Fisher (A14527), were cultured in serum-free SMM-293TII media (Sino Biological M293TII) at 37 °C in a humidified, 5% CO₂ atmosphere incubator, shaking at 150 rpm. HEK293 (ATCC CRL-1573) and HEK293T (ATCC CRL-3216) cells obtained from ATCC were cultured at 37 °C with 5% CO₂ in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.