Generation and Purification of Chimeric Monoclonal Antibodies

Human-mouse chimeric MAb ch61 was generated and purified from culture supernatants as described previously [23] (link). Briefly, total RNA was extracted from mouse hybridoma cells producing MAb m61, and the variable heavy- and light-chain regions were amplified by RT-PCR with primers designed for the antibodies. The PCR products were cloned into an expression vector. Stable cell lines expressing recombinant MAb ch61 were obtained by transfection of CHO DG44 cells (Invitrogen, Carlsbad, CA). Chimeric MAbs (ch133 and ch226) specific for the Ebola virus glycoprotein were generated as control MAbs using the same methodology [23] (link). These human-mouse chimeric MAbs were purified from culture supernatants using rProtein A Sepharose Fast Flow (GE Healthcare) and EndoTrap red (Profos AG). MAb purity (>98%) and endotoxin levels (<1.0 EU/ml) were confirmed by performing SDS-PAGE and with an Endospecy ES-50M kit (Seikagaku Corporation), respectively.