Characterization of Neospora caninum iscom antigens using monoclonal antibodies

Neospora caninum is a cyst-forming coccidian parasite which in cattle can cause abortion and birth of feeble calves. For the serological diagnosis of neosporosis, antibodies directed to the parasite can be demonstrated by ELISA, utilizing N. caninum proteins incorporated into iscoms (immunostimulating complexes) as antigen. Electrophoretic and immunoblotting studies had earlier revealed that N. caninum iscoms contained a restricted number of proteins compared with soluble parasite extracts, but the cellular origin of the incorporated proteins has not yet been determined. In the present study, monoclonal antibodies were raised against N. caninum iscoms. Six of these, named Ncmab-4, 7, 10, 13, 17 and 24, were used to characterize the N. caninum iscom antigen. The nature of the reactive epitopes and their localization within N. caninum tachyzoites were determined by means of immunological methods, including immunoblot, IFAT and immunogold electron microscopy. The apparent molecular weights of the dominant iscom antigens were found to be 18, 30, 32, 41 and 61 kDa. While the 61 kDa antigen was located intracellularly, the others were found on the parasite surface as well as within distinct intracellular compartments.