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, overlapping with the V-gene proximal region of the mouse CH1 and Ck regions, respectively) to allow cloning of the V-genes. Synthesized DNA fragments were inserted into corresponding pHFB-IgG1 and pHFB-k vectors (HiFiBiO heavy and light chain expression plasmids) in frame with the human IgG1 or kappa constant regions by In-Fusion DNA assembly according to manufacturer's instructions, and transformed into E. coli Stellar Chemical Competent Cells (Clontech)
, Correctly cloned VH-VL pairs were co-transfected into serum-free Expi293F suspension cell cultures (Life Technologies) using ExpiFectamine reagents (Life Technologies) following the manufacturer's instructions. Transfection reactions were incubated for 4 days, at which point supernatants were collected, clarified by centrifugation at 1400 rpm for 15 min at RT, and stored at 4°C. For affinity measurements
, After purification, IgGs were desalted with HiTrap Desalting Column, HiTrap Protein G Column (GE Healthcare)
, Maxisorp plates were coated with 100 µL of 1 µg/ml TT or 5 µg/ml GPI in PBS overnight at 4?. Plates were blocked with PBS containing 1%. BSA. Naive, immune mice serums, IgGs from culture supernatants or control IgG at at 1 µg/ml were diluted in series. Anti-human Fc-HRP at 0.05 µg/mL (Bethyl) was used for detection. The assay was revealed with OPD, p.2
, Affinities of the recombinant anti-TT antibodies identified by CelliGO: EC50s for anti-TT
, Pall ForteBio) was used to quantify antibody concentrations in supernatants of transfected Expi293F cells, and to measure affinities of the recombinant mouse-human chimeric anti-GPI antibodies. For the latter, biosensors were loaded with 1 ug/mL antibody, association and dissociation signals (10 min per step) were measured against 5 different concentrations of GPI (1.85, 5.5, 16.6, 50 and 150 nM), Bio-layer interferometry and real-time surface plasmon resonance. Affinities of the recombinant anti-GPI antibodies identified by CelliGO: Bio-layer interferometry with an anti-human IgG sensor on Octet systems
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