Characterization of Murine Monoclonal Antibodies by Hybridomatechnology for the Peste Des Petits Ruminants (PPRV)Vaccine

Abstract

Peste des petites ruminants (PPRV) is an acute and highly contagious viral disease of sheep and goats. It continues to be a major cause of economic losses worldwide. The causative agent is from the genus Morbillivirus. It is very much antigenically similar to the rinderpest virus. In this study, we developed specific monoclonal antibodies (mAbs) against PPRV. These monoclonal antibodies will be used for the future development of diagnostic assays against PPRV. Monoclonal antibodies are widely used in molecular biology and biomedical research. However, the generation of these molecules is complicated and time-consuming. The production of PPRV-specific mAbs involved immunizing BALB/c mice with a commercially available PPRV vaccine. Spleenocytes were then fused with myeloma cells (SP2/0) using polyethylene glycol fusion (PEG fusion). A total of five monoclonal antibodies (mAbs) out of seven were found positive in IELISA These are 1A-1, 2A-5, 3B-5, 4C-2, and 5C-5 raised against the PPRV vaccine. These were developed by the CBIS method (cell-based immunization and screening), that is fusion of myeloma cell line SP2/0 with mice spleenocytes, collected from purified PPRV vaccine immunized mice. These mAbs were then characterized by Indirect ELISA and western blot analysis(SDS-PAGE). One of the positive hybridoma clones (3B-5) secreting mAbs belonged to the IgG class, which was purified using a Protein A- Plus spin kit (Thermo Scientific) and showed a single band in SDS-PAGE. The mAbs named, (3B-5) represented a positive, 70-kDa protein band in SDS-PAGE analysis. The mAbs established in this study are useful for studying the interaction between PPRV and its target cell. The current study effectively characterized the mAbs and found one positive 3B-5 was proved to be efficient for detecting antibodies against PPRV in sheep and goat sera and would use to develop diagnostic assays for PPRV in Pakistan. This is the first ever report from Pakistan, that evaluate the practical approach for the characterization of monoclonal antibodies against the PPRV virus.