Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

Han, Baek-Sang; Jang, Ho-Young; Racine, Trina; Qiu, Xiangguo; Sin, Jeong-Im

Hybridoma cells were cultured in cDMEM containing HT. The cell supernatants were collected and used for purification of MAbs. MAbs (IgG) were purified using the protein G resin column in accordance with the manufacturer's protocol (Peptron, Daejon, Korea). Briefly, the protein G resin column was pre-equilibrated with 10 mL binding buffer (0.1 M sodium phosphate buffer, 0.15 M NaCl, pH 7.4). Samples were filtered through a 0.2 μm filter and mixed with an equal volume of binding buffer. The mixed samples were loaded onto the resin column, followed by 2 washes with 10 mL of binding buffer. Finally, the bound antibodies were eluted by adding elution buffer (0.2 M glycine buffer, pH 2.5). In particular, 0.5 mL of each eluted sample fraction was added with 65 μL of neutralization buffer (1 M Tris/HCl buffer, pH 9.0) to adjust pH. Finally, the antibodies were dialyzed in PBS.