Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

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

Vero E6 cells were cultured in each well of a 96-well tissue culture plate. The following day, when the cells were approxi- The G418-selected cell colonies (HEK293-1, 2, 3, 4, 5, 6) were reacted with 2 µL of GP-specific immune sera, followed by reaction with FITC-conjugated anti-mouse IgG (whole molecules) for flow cytometry. The cells were measured for reactivity to GP expressed on HEK293 cells. (B) HEK293-5 cells which prominently reacted with anti-GP sera were further cultured and then the cell lysates were run on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane and reacted with commercial anti-GP antibodies for Western blot assay. (C) Mice were injected by IM-EP with pcDNA3-GP (50 µg/mouse) at 0 and 4 weeks. The mice were bled at 2 weeks following the final injection and sera were collected. (C) The sera were reacted with 5×10 5 HEK293-GP and control HEK293 cells, followed by incubating with FITC-conjugated anti-mouse IgG (Fc) for flow cytometry. GP, glycoprotein; FITC, fluorescein isothiocyanate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IM, intramuscular; EP, electroporation. mately 80% confluent, 100 µg/mL of each of the 5 antibodies, as well as 2G4 used as a positive control for neutralization [6] , were serially diluted in cDMEM. To this, an equal volume of Ebola virus Makona (GenBank No. KJ660348.2) was added so that each well would receive 100 plaque forming units. The virus-antibody mixture was incubated for 1 hour and then 100 µL of the virus-antibody mixture was added to the 96-well. After incubating the 96-well plate for 1 hour at 37°C, the mixture was removed and 200 µL of 2% FBS DMEM was added to each well. The plate was returned to the 37°C incubator and plates were read for cytopathic effect 13-day post-infection. This assay was performed in the biosafety level 4 laboratory at the National Microbiology Laboratory, part of the public Health Agency of Canada in Winnipeg, Manitoba, Canada.