Purification of Ig proteins

O2F3 (mouse IgM) was purified from the culture supernatant of O2F3 hybridoma cells cultured in RPMI 1640 media supplemented with 10% FBS for 7 days at 5% CO₂ and 37 °C. O2F3 IgM protein was purified from the culture supernatant by affinity chromatography using agarose-goat anti-mouse IgM/μ chain-specific resin (Sigma-Aldrich; cat# A4540) according to the manufacturer’s instructions.

ER-directed chimeric 3D8 IgG1 protein was purified from the culture supernatant of FreeStyle 293-F cells (ThermoFisher; cat# 12338). FreeStyle 293-F cells (100 ml) at a cell density of 1 × 10⁶ cells/ml were seeded 24 hours prior to transfection in a 500 ml flask (Corning; cat# 431145) to ensure they reached the appropriate cell density (2 × 10⁶ cells/ml) at the time of transfection. Culturing was performed in serum-free FreeStyle 293 medium (Invitrogen; cat# 12338) at 8% CO₂ and 37 °C with shaking at 130 rpm. Plasmid (200 μg) encoding a pair of Ig H and L chains was transiently introduced into the 100 ml FreeStyle 293-F cell culture (2 × 10⁶ cells/ml) with PEI reagent (25 kDa, Polyscience; cat# 23966-2). Specifically, PEI reagent (400 μg) was incubated with plasmid DNA (200 μg) at room temperature for 10 min, and mixtures were inoculated into 100 ml of cells to achieve a 4 μg/ml final PEI concentration. After 6–7 days, the culture supernatant was harvested by centrifugation, and Ig proteins were purified by affinity chromatography using agarose-Protein A (GE Healthcare; cat# 17-1279-02) according to the manufacturer’s instructions.