Generation of monoclonal antibodies and epitope mapping of ApxIVA of Actinobacillus pleuropneumoniae

To study functions of ApxIV, a species-specific and in vivo inducible RTX toxin identified in Actinobacillus pleuropneumoniae recently, and to develop a diagnostic trial distinguishing the pigs infected naturally and vaccinated with inactivated and/or subunit vaccines, we attempted to prepare monoclonal antibodies against ApxIV. BALB/c mice were immunized with ApxIVAN and ApxIVAC which are N- and C-terminal halvies (814 and 997 amino acids, respectively) of ApxIVA produced in E. coli BL21 (DE3), respectively. Eight monoclonal antibodies were selected, four (designated as 1A8, 1G5, 3E7 and 4H9) against ApxIVAN and another four (named as 1B12, 2E5, 4D8 and 4G2) against ApxIVAC. Western blot and ELISA additivity assays suggested that all monoclonal antibodies except 1A8 are specific to the corresponding immunogen, 1A8 reacts with both immunogens which have a overlapping region of 156 residues. ELISA additivity tests revealed that at least five epitopes in ApxIV are defined by eight monoclonal antibodies, two between 1 and 866 amino acids, one between 867 and 1022 amino acids and two between 1023 and 1863 amino acids. In conclusion, we have succeeded in producing eight monoclonal antibodies, which react with five different epitopes of ApxIV.