Unique scFv clones were converted into human IgG1 using mixed universal primers with degeneracy [27 (link)]. Individual heavy and light variable chains were amplified using PrimeStar GXL polymerase (Takara Bio). Gel-purified variable chain fragments were cloned into digested vectors using In-fusion HD cloning enzyme mix (Takara Bio). After the converted plasmid was sequenced, sequences verified IgG plasmids were transfected into Expi 293 cells at the 2-mL scale. After culturing for 5 days, cells were removed and antibody-containing supernatant was collected for screening assay.
For milligram-scale antibody purification, Expi293-produced antibodies were purified using CaptivA Protein A affinity resin (Repligen) and eluted with 0.1 M glycine (pH = 2.5) and then neutralized with 1/20 volume 1 M Tris-HCl (pH = 9). Buffer exchange to DPBS was done using Amicon Ultra-15 ultrafiltration units (Mw cutoff = 30 k) (MilliporeSigma).
For milligram-scale antibody purification, Expi293-produced antibodies were purified using CaptivA Protein A affinity resin (Repligen) and eluted with 0.1 M glycine (pH = 2.5) and then neutralized with 1/20 volume 1 M Tris-HCl (pH = 9). Buffer exchange to DPBS was done using Amicon Ultra-15 ultrafiltration units (Mw cutoff = 30 k) (MilliporeSigma).
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