Characterization of monoclonal antibodies against antigenic epitopes of Actinobacillus pleuropneumoniae ApxIVA

Abstract

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is a highly contagious disease that leads to enormous economic losses in pig industry, worldwide. Of the many virulence factors produced by the causative bacterium, ApxA exotoxins have been considered as the most important contributor to the disease. The toxins are classified into four different types; ApxIA, ApxIIA, ApxIIIA and ApxIVA. Uniquely, ApxIVA is expressed across all serotypes of A. pleuropneumoniae only during in vivo infection in pigs. Active research focusing on resolving the precious roles and mechanisms of the toxins is still at its primitive stage. In this study, we report the development of monoclonal antibodies against the two major antigenic epitopes that were characterized in our previous study incorporating the in silico predictions and protein modeling analyses. Recombinant proteins of the selected epitopes were expressed and purified after molecular cloning of the corresponding partial genes in E. coli expression system. Subsequently, we generated hybridomas with lymphoid cells from the rats immunized with the recombinantly expressed proteins of Apx. Consequently, hybridomas exhibiting strong productivity of the monoclonal antibodies were selected for downstream verifications that tested for reactivity and specificity using Western blot and ELISA. Our results strongly suggest the potential application of the monoclonal antibodies developed in this study as useful reagents to further elaborate the mechanism of the A. pleuropneumoniae infection in pig.