Vivacell device

Antibodies were purified from a guinea pig (immunized with lysate of Vero cells) serum using protein A HiTrap MabSelect Xtra column (1 mL; GE Healthcare, Chicago, IL, USA). Serum was 2-fold diluted with the binding buffer (20 mM phosphate buffer, 0.15 M NaCl, pH 7.2) and loaded (2 mL/run) to the pre-equilibrated protein A column at a flow rate of 2 mL/min on an ÄKTApurifier 100 system equipped with P-900, UV-900, and pH/C-900 (GE Healthcare, USA) at room temperature (RT). The absorbance was monitored at 280 nm. The bound antibodies were eluted from the column with 20 mM glycine, 0.15 M NaCl, and pH 2.3. Eluted fractions from all runs were pooled, concentrated, and transferred to the binding buffer using a Vivacell device (Sartorius, Göttingen, Germany) with a 50 kDa molecular weight cut-off (MWCO) polyethersulfone membrane, resulting in polyclonal anti-Vero IgG sample that was used as a reagent for preparation of the IgG-HRP conjugate and for plate coating in the ELISA.

In preliminary experiment CCP, aliquoted and stored at -20 °C until use, was thawed at room temperature and incubated at 56 °C for 1 h in a thermomixer (Eppendorf, Germany). After centrifugation at 3,200 × g for 45 min and discarding of the pellet, caprylic acid was added in a dropwise manner so that final concentrations of 1-7% (V/V) in 2-fold diluted reaction mixtures (V = 1 mL) were prepared, each in duplicate. Saline was used as diluent. Precipitation was performed by vigorous stirring (850 rpm) at 23 °C for 1 h, followed by centrifugation (3,200 × g, 45 min). Minimal caprylic acid concentration giving the highest IgG purity of acceptable yield was chosen as the most beneficial, as described in Results. In all subsequent experiments the precipitation step was performed under optimized conditions. Furthermore, the whole procedure was scaled up 40-fold. IgG-containing supernatant was first filtered through a hydrophilic cloth and a cellulose acetate filter with a pore size of 5 μm (Sartorius, Germany), and afterwards diafiltrated into 20 mM sodium acetate buffer, pH 5.0 using a Vivacell device (Sartorius, Germany) with a 100 kDa molecular weight cut-off (MWCO) polyethersulfone membrane. Several repeated cycles of concentration and dilution were performed to achieve approximately 4 logs of matrix exchange.