Purification of Mouse and Chimeric Antibodies

To prepare mouse Ab proteins, mouse hybridoma cells were cultured in RPMI1640 media (ThermoFisher Scientific; cat# 11875-093) supplemented with 10% fetal bovine serum (FBS) at 37 °C in an atmosphere of 5% CO₂. Mouse 3D8 IgG2a/κ and 6C407 IgG2a/κ were purified from the supernatant of hybridoma cells, and 3D8 IgG and 6C407 IgG were purified by affinity chromatography using Protein L-agarose resin that binds to the Vκ region of Abs (GE Healthcare; cat# 17-5478-01). Chimeric Ab proteins were prepared from cultures of FreeStyle 293-F serum-free and suspension-adapted HEK293F cells (ThermoFisher Scientific; cat# R79007). HEK293F cells were cultured in serum-free FreeStyle 293 media (ThermoFisher Scientific; cat# 12338018) with 8% CO₂ and shaking at 130 rpm in the 37 °C incubator. FreeStyle 293-F cells (100 ml) at a density of 2 × 10⁶ cells/ml were transfected with 200 μg of KV10 plasmid encoding an Ab gene using 400 μg of polyethylenimine (PEI) reagent with a molecular weight of ~25 kDa (Polyscience; cat# 23966-2), a final PEI concentration of 4 μg/ml^{46 (link)}. After 7 days, the culture supernatants were harvested by centrifugation, and two chimeric MC-IgY and two chimeric MH-IgG proteins were purified by affinity chromatography using Protein L (GE Healthcare; cat# 17-5478-01) and Protein A column (GE Healthcare; cat# 17-1279-01), respectively.