Production and characterization of a monoclonal antibody specific to 16 kDa antigen of Paramphistomum gracile

A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.