4.1. ScFv-h3D6 Production

ScFv-h3D6 was produced as previously described [18]. Briefly, the scFv-h3D6 gene was cloned into the pPicZαA vector (Invitrogen, Carlsbad, CA, USA) and expressed in KM71H P. pastoris cells. Cells were grown until an OD₆₀₀ of 2.0 was reached. Then, methanol was supplemented every 24 h to induce protein expression. After 48 h of expression, cells were centrifuged at 3000× g and 4 °C for 10 min. ScFv-h3D6 was precipitated with ammonium sulfate. After centrifugation at 100,000× g and 4 °C for 1 h, the pellet was re-suspended in 10 mM Na₂HPO₄ (pH 6.5) and double dialyzed. Finally, cationic exchange chromatography was performed in a Resource S column coupled to a UP10 AKTA (GE Healthcare, Chicago, IL, USA). Pure scFv-h3D6 was dialyzed to PBS (pH 7.4) and stored at −20 °C until further use.