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 cell supernatants or antibody samples were separated on a 12% SDS-polyacrylamide gel. The proteins on the gel were analyzed by staining with the brilliant blue R staining solution (Elpis-Biotech, Daejon, Korea). The proteins were also electrophoretically transferred to Immobilin-P membranes (Merck Millipore). The membrane was pre-equilibrated with TBST solution (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk for 1 hour. For detection of antibodies, the membrane was incubated with HRP-conjugated anti-mouse IgG (H+L) (Komabiotech) for 1 hour at room temperature. In each step, the membrane was washed 3 times with TBST. The immunoreactive protein bands were visualized using the ECL detection reagents (Phile-Korea, Seoul, Korea). In one case, HEK293-GP cell lystes were separated on a 10% SDS-polyacrylamide gel, and transblotted to Immobilin-P membranes. The membrane was cut into many strips, which were reacted with each of the purified IgG and, as a control, anti-Ebola virus GP commercial antibodies raised in rabbits (Sino Biologicals, Beijing, China). This was followed by incubation with HRP-conjugated anti-mouse IgG http://www.ecevr.org/ https://doi.org/10.7774/cevr.2018.7.2.119 (H+L) or HRP-conjugated anti-rabbit IgG (Komabiotech) for Western blot assay.