Production and Purification of 10E8 Antibody

Transient expression of the 10E8 antibody was undertaken in 293F cells or 293 Expi cells per the manufacturer’s instructions (Life Technologies) by co-transfection of equal amounts of the 10E8 heavy and light chain plasmids, as previously described^{66 (link)}. 10E8 IgG was purified by capture with Protein A sepharose (Pierce) followed by elution in low pH (Pierce) with adjustment of eluate pH with Tris-Cl pH 8.0. The 10E8 Fab was prepared using endoproteinase Lys-C (Roche) digestion, as described^{66 (link)}. The LysC protease was added at concentrations of 1:1,000 to 1:10,000 and the digestion undertaken at 37°C for 4–6 h. Digestion reactions were stopped with protease inhibitor tablets (Roche) and cleaved products were run over a Protein A column to segregate the Fc fragment. 10E8 Fab was then subjected to cation exchange (Mono S) and size-exclusion (S200) chromatography, followed by dialysis of peak fractions into 150 mM NaCl, 2.5 mM Tris, pH 7.5, 0.02% NaN₃.