Generating Anti-mouse C5 Chimeric Antibody

Total RNA was prepared using TRI reagent (Invitrogen) form spleen, bone marrow and bursa of Fabricius of white leghorn chickens which had been immunized against MG4 domain and β-chain of mouse C5. First-strand cDNA was synthesized using superscript reverse transcriptase with oligo (dT) priming (Invitrogen). Using this cDNA, a phage-display library of rabbit single-chain variable fragment (scFv) was constructed using pComb3XSS phagemid vector as previously described [46 , 47 (link)]. After the library construction, scFv clones were selected from the library through five rounds of biopanning as described previously [46 ]. For each round of biopanning, 1.5 μg of mouse MG4 domain and β-chain protein coated magnetic beads (Dynabeads M-270 Epoxy; Invitrogen) were used. The anti-mouse C5 antibody is chimeric antibody containing chicken scFv and murine IgG1 of which heavy chain and light chain were subcloned into the pCT184 and pCT146, respectively. These vectors were co-transfected to Chinese hamster ovary-K1 cells and the supernatant was subjected to protein G affinity gel chromatography for purification. This process of antibody production was supported by Celltrion.