[Preparation of mouse monoclonal antibodies against EV71 capsid protein VP1]

Objective To prepare enterovirus 71 (EV71) capsid protein VP1-specific murine monoclonal antibodies (mAbs) and determine their characteristics. Methods We synthesized EV71 VP1-encoding gene and cloned it into prokaryotic plasmid pET21a. Recombinant VP1 was used to immunize BALB/c mice; the splenocytes were fused with myeloma SP2/0 cell line and the hybridoma cells which could produce VP1-specific mAbs were screened using ELISA; the hybridoma cells were inoculated intraperitoneally into mice. VP1-specific mAbs were purified from ascites using protein G affinity chromatography, and mAb purifiy and specificity were detected by SDS-PAGE and Western blotting, respectively. Based on EV71-infected Vero cells, Western blotting and indirect immunofluorescence assay (IFA) were used to assess the ability of VP1-specific mAbs to recognize EV71. Results EV71-VP1 was successfully expressed and four VP1-specific mAb-producing hybridoma cells were generated (6E, 8D, 9F and 10C). Finally, mouse mAbs secreted by the hybridoma cells recognized EV71. Conclusion Four EV71-VP1-specific murine mAbs have been developed.