The total proteins of plain E.coli(DH5 ?)were coupled to the Sephacryl S300 oxidized with periodate,and the preliminarily purified antiserum was loaded repeatedly on the column for 18 hours.The unspecific anti\|E.coli antibodies would be absorbed and the antibodies would be purified.It was more specific to do western blot using antibodies treated by this method than using ones without treatment.

PURPOSE: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. MATERIALS AND METHODS: For 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. RESULTS: Twelve 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. CONCLUSION: This 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.
Animals ; Antibodies* ; Antibody Formation ; Blotting, Western ; Clone Cells ; Ebolavirus* ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Glycoproteins* ; Hemorrhagic Fever, Ebola ; Hybridomas ; Immunoglobulin G* ; In Vitro Techniques ; Mice ; Sensitivity and Specificity ; Spleen ; Vaccines ; Vaccines, DNA

Ebolavirus ; pathogenicity ; Endemic Diseases ; Hemorrhagic Fever, Ebola ; epidemiology ; virology ; Humans