Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

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

In the current study, we successfully generated IgG-secreting hybridomas by boosting the GP DNA vaccine-primed mice with GP emulsified in incomplete Freund's adjuvant and finally with GP dissolved in PBS. This result corroborates the notion that proteins, as a booster vaccine type, may play a critical role in boosting the production of IgG-secreting hybridomas in the DNA vaccine platform. In the screening scheme using ELISA and Western blot assay, we were able to obtain a total of 12 polyclonal hybridomas secreting GP-specific IgG types. Among these, we obtained five clones (producing an IgG isotype) and two clones (producing an IgM isotype) by the limiting dilution sub-culture method. The purified monoclonal IgG antibodies from five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) reacted with Ebola virus GP, but not MERS coronavirus Spike protein and SFTS virus-GP, suggesting that they are indeed antigenspecific. Moreover, they were IgG kappa types and remained reactive to Ebola virus GP at the lowest concentrations ranging from 1.593 to 7.8 ng/mL. In particular, the IgG antibodies from 3 clones (D11-3, D12-1 and E140-2) recognized GP expressed on the cell surface of HEK293-GP, suggesting that they may recognize the native and outer-membrane forms of GP. On the other hand, the IgG antibodies from one clone (D12-1) recognized GP1e (containing mucin-like region) alone, suggesting that this IgG antibody recognizes the mucin-like region of GP. However, the IgG antibodies from four clones (C36-1, D11-3, D34-2, and E140-2) recognized none of the http://www.ecevr.org/ https://doi.org/10.7774/cevr.2018.7.2.119 five recombinant GP1s, suggesting that the four purified IgG antibodies may recognize the tertiary forms of GP and/or other GP sites excluding the mucin-like region and receptorbinding domain of GP. Moreover, none of the five purified IgG antibodies recognized the denatured forms of GP. These data are in line with our observation that sera from animals immunized with GP DNA vaccines were unable to recognize the denatured forms of GP by Western blot assay (data not included). Taken together, these data show that the five purified IgG antibodies can recognize only a native from of GP. Here, it is notable that the remaining five polyclonal hybridomas (E2, E8, E22, E31, and E60) appear to lose their ability to produce IgG antibodies after continuous cell culture and passage, leading to a failure in making an IgG producer. Among numerous single clones obtained after limiting dilution subculture from the polyclonal hybridomas, not a single clone was observed to secrete Ag-specific IgG antibodies (data not included). In this context, it is highly likely that the loss of IgG productivity might be ascribed to a decrease in the fraction of IgG-producing hybridoma cells over time, possibly resulting from the loss of the chromosomes containing the gene loci for the IgG heavy and light chains. This is supported by the previous finding of others [13] . On the other hand, clones E12-10 and E49-3, displaying the highest OD values among numerous clones, were secreted as an IgM producer. Prior to limiting dilution sub-culture, two hybridomas (E12 and E49) generated IgG types, but later lost their ability to generate IgG antibodies. It is likely that in the case of 2 hybridomas (E12 and E49), IgG-producing cell populations may have disappeared and then been replaced by IgM-producing cell populations. This notion is supported by our observation that the cel