Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

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

We previously reported that GP DNA vaccines induce Ag-specific antibody responses [10] . In this study, we investigated whether GP DNA vaccines might induce IgG responses to the native forms of GP expressed on the cell surface. Therefore, HEK293 cells were transfected with pcDNA3-GP and then selected against G418. Cells from the six G418-selected cell colonies were reacted with GP-specific immune sera [10] , followed by flow cytometry using FITC-conjugated anti-mouse IgG (whole molecule) as the secondary Ab (Fig. 1A) . The HEK-293-5 cells, which displayed the prominent binding reactivity to the immune sera, were chosen and tested again for their GP expression status using Western blot assay (Fig. 1B) . As seen in Fig. 1B , HEK293-5 displayed 160-140 kDa GP bands, as opposed to control HEK293 cells. This cell was designated HEK293-GP. We next immunized mice by IM-EP with pcD-NA3-GP at 0 and 4 weeks and then bled at 6 weeks. The sera were collected and reacted with HEK293-GP cells. As shown in Fig. 1C , the immune sera reacted with HEK293-GP cells, but not with HEK293 cells (without GP expression) by flow cytometry using FITC-conjugated anti-mouse IgG (Fc) as the secondary Ab. This result shows that GP DNA vaccines can indeed induce IgG antibodies that recognize the native forms of GP expressed on the cell surface in vivo.