Production and characterization of monoclonal antibody against Perkinsus olseni in undulated surf clams Paphia undulata

Abstract

Perkinsosis is a mollusk disease caused by protozoan parasite belonging to the genus Perkinsus. Perkinsosis has been found in some commercially important mollusks including oysters, clams and abalones. Heavy infection with Perkinsus often results in tissue inflammation and mass mortalities. To date, three stages have been described in the pathogen’s life cycle: trophozoite, hypnospore and zoospore. The infections in mollusks cannot be diagnosed without specialized testing. Various methods have been applied in the diagnosis of these infections including the fluid thioglycollate medium (FTM) technique, immunological and PCR assays. In this study, monoclonal antibodies (MAb) for immunological assay against Perkinsus olseni were produced and characterized. Three ICR mice were immunized with P. olseni in the zoospore stage. The polyclonal antiserum showed the antibody titer of 1:128,000 by dot blotting. After three fusions of the splenocytes and myeloma cells, two hybridoma clones producing MAbs (2/B4/A2 and 8/H11/F2) against zoopores were obtained. The isotype of the MAbs 2/B4/A2 was IgG1 while that of 8/H11/F2 was IgM subclass. Only MAb 8/H11/F2 showed immunoreactivity with the zoospores and hypnospores stage. Both MAbs can be used to identify zoospores P. olseni from clams by dot blotting with the sensitivity range of 10⁸–10⁹ cell/ml.