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KMID : 1039120170060020135
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Clinical and Experimental Vaccine Research
2017 Volume.6 No. 2 p.135 ~ p.145
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Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production
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Lee Si-Hyeong
Han Baek-Sang
Choe Jong-Seon
Sin Jeong-Im
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Abstract
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Purpose: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies.
Materials and Methods: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells.
Results: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone.
Conclusion: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.
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KEYWORD
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Ebola virus, Hybridoma, DNA vaccines, Glycoprotein, Antibody production
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