Clin Exp Vaccine Res.  2018 Jul;7(2):119-128. 10.7774/cevr.2018.7.2.119.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

• Han BS
• Jang HY
• Racine T
• Qiu X
• Sin JI

• KMID: 2418090
• DOI: http://doi.org/10.7774/cevr.2018.7.2.119

Abstract

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.

Keyword

Ebolavirus; Hybridomas; DNA vaccines; Glycoproteins; Antibody formation; Neutralization

MeSH Terms

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
Antibodies
Glycoproteins
Immunoglobulin G
Vaccines
Vaccines, DNA

Figure

• 

  Fig. 1 Stable transfection of HEK293 cells with pcDNA3-GP and selection of GP-expressing HEK293 cells (A, B), and evaluation of sera obtained after GP DNA vaccination for their ability to recognize GP expressed on the cell surface. (A) HEK293 cells were stably transfected with pcDNA3-GP. 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 5×105 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.

• 

  Fig. 2 Purification of the IgG antibodies from the five hybridoma clones, and the evaluation of their Ag-specific binding activity. (A) The hybridomas (D36, D11, D12, D34, and E140) were diluted to make a single cell by the limiting dilution sub-clone method. The resulting monoclonal hybridoma clones were designated C36-1, D11-3, D12-1, and E140-2. IgG antibodies were purified from the cell supernatants using the protein G column. Two µg of the purified IgG antibodies were run on a 12% SDS-PAGE for brilliant blue R staining. (B) The purified IgG (2 µg/mL) were reacted with Ebola virus GP, MERS coronavirus spike protein and SFTS virus GP for ELISA. (C) Evaluation of the lowest GP-binding concentration of IgG antibodies. Two micrograms per milliliter of IgG antibodies from the clones, C36-1, D11-3, D12-1, and E140-2 were serially diluted by 2-fold and then reacted with GP in parallel with control IgG (2 µg/mL) to determine the lowest GP-binding concentration using ELISA. (D) Five recombinant GP1 proteins (1 µg/mL) were coated and reacted with the five purified IgG antibodies (0.2 µg/mL) from clones, C36-1, D11-3, D12-1, and E140-2 for ELISA. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel; GP, glycoprotein; MERS, Middle East respiratory syndrome; SFTS, severe fever with thrombocytopenia syndrome; ELISA, enzyme-linked immunosorbent assay; OD, optical density.

• 

  Fig. 3 Evaluation of the five purified IgG antibodies for their binding activity to the native forms of GP expressed on the cell surface. One microgram per mililiter of the IgG antibodies from clones, D36-1, D11-3, D12-1, D34-2, and E140-2 were reacted with 5×105 HEK293-GP and HEK293 cells, followed by incubation with FITC-conjugated anti-mouse IgG (Fc) for flow cytometry. Thin line: control IgG, Thick line: experimental IgG. GP, glycoprotein; FITC, fluorescein isothiocyanate.

• 

  Fig. 4 Binding activity of the five purified IgG antibodies to the denatured forms of GP by Western blot assay. Thirty micrograms of HEK293-GP cell lysates were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins on the gel were transferred to a nitrocellulose membrane. The membrane was cut into strips, which were reacted with each of the five purified IgG antibodies (1 µg/mL) or commercial anti-Ebola virus GP, followed by Western blot assay. Arrows indicates Ebola virus GP. GP, glycoprotein.

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