Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

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

Each well of a 96 well plate was coated at 4°C overnight with recombinant GP and GP1 proteins (0.5 µg/mL in PBS). The next day the plate was washed 3 times with washing buffer (PBS+0.05% Tween 20) and then blocked for 1 hour with blocking buffer (washing buffer+2% bovine serum albumin). Then, the plate was reacted with test samples for 1 hour. After this, the plates were washed 3 times with washing buffer and reacted for 1 hour with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (H [heavy]+L [light]) diluted in blocking buffer. After washing the plate, an ABTS solution (Merck Millipore, Billerica, MA, USA) was added to each well. Optical density (OD) was read at 405 nm using an ELISA reader. In particular, for the determination of the relative levels of GPspecific IgG subclasses, HRP-conjugated anti-murine IgG1, IgG2a, IgG2b and IgG3 antibodies (Thermo Scientific, Waltham, MA, USA) were substituted for HRP-conjugated anti-murine IgG (H+L). HRP-conjugated anti-mouse IgG (H+L) was purchased from Komabiotech (Seoul, Korea).