Production of Monoclonal and Polyclonal Antibodies Against PRRSV MLVD23-S tag Peptide Sequence

Katelyn Bettin, South Dakota State University


Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus which belongs to the family Arteriviridae. Since its emergence in the late 1980’s and early 1990’s, PRRSV has had a major economic effect on the swine industry. The most notable effects of this disease are respiratory distress in piglets and late-term pregnancy failures in sows. To track and control spread of this disease, the industry must be able determine which animals have been naturally infected with PRRSV as this contributes to major disease outbreaks and localized pandemics. While vaccines help control the spread of a disease, they also interfere with tracking of disease outbreaks. A DIVA test allows for the differentiation of animals which were naturally infected from those that were infected via immunization and is used in conjunction with a vaccine. Our primary objective was to develop rabbit polyclonal and mouse monoclonal antibodies against a highly immunogenic, heterologous gene not native to PRRSV to be used as reagents for a PRRSV specific DIVA (Differentiating Infected from Vaccinated Animals) test. To start the project, non-structural protein PRRSV-NSP2 was modified to include an immune marker (S-tag genetic marker) to be used as a positive selection tag that allows for the diagnostic detection of the vaccine virus strain. This protein was cloned into a plasmid expression vector, expressed in E. coli bacteria, and purified in order to be used as an antigen to immunize mice and rabbits. The immunization of rabbits was effective for producing polyclonal antibodies to be used for further research and diagnostic purposes, while the immunization of the mice was used to generate monoclonal antibodies through the formation of hybridoma cells. A hybridoma is produced by fusing splenic B cells from the immunized mice with mouse tumor cells (NS-1 myeloma). These hybridomas produced monoclonal antibodies which were expected to be specific to PRRSV MLVΔ23-S tag epitopes. These monoclonal antibodies were characterized and their ability to recognize PRRSV MLVΔ23-S tag epitopes was assessed via enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and Western blot assay. Overall, we recovered eighteen separate hybridoma cell lines which were immunoreactive via ELISA and Western blotting. Eight of these were also immunoreactive via IFA. We also obtained approximately 75 mL of anti-PRRSV S-tag specific rabbit polyclonal antisera. These resulting monoclonal and polyclonal antibodies will be useful to develop a DIVA test for the veterinary diagnostic industry. This test can be used to monitor the effectiveness of the PRRSV vaccine and spread of the disease in order to assist in reducing the major economic impact of PRRSV on the swine industry.