Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

Abstract

PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and MethodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Dual monoclonal antibody-based sandwich ELISA for detection of in vitro packaged Ebola virus

“…Qiu et al, used recombinant VZV-Ebola GP, followed by GP protein to immune mouse, achieved several cell lines against Zaire Ebola virus, targeting different subunits (GP1 or GP2), without further analysis about antibody properties [20]. Han et al, utilized pCAGGS-GP and GP protein to immune mouse and obtain several mice, however they did not provide detailed information of the GP-specific mAbs [26]. Here we applied the recombinant GP to immunize mice and achieved several mAbs, furthermore we identified the mAbs in multiple aspects, including binding activities, affinities, accurate epitopes.…”

CD8+ T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection

“…into mice on the indicated days. A hybridoma cell line (clone 2.43) was purchased from the American Type Culture Collection (Manassas, VA, USA), and anti-CD8 IgGs were purified as previously described ( 22 ). Control IgGs were purchased from Sigma-Aldrich (St. Louis, MO, USA).…”