28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Development And Characterization Of Monoclonal Antibodies Generated Against The Extracellular Products Of The Oyster Parasite Perkinsus marinus Christopher G. Earnhart and Stephen L. Kaattari Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA 23062 Extracellular products (ECP) of the oyster parasite Perkinsus marinus may contain parasite virulence factors.  To study the induction, secretion levels, and release mechanism of these factors both in vitro and in vivo, a panel of monoclonal antibodies was produced that could recognize the ECP.  Mice were immunized with ultrafiltration-concentrated supernatants of P. marinus grown in a protein-free, defined medium.  This preparation was poorly immunogenic and toxic to experimental animals.  Neither heat denaturation nor inhibition of serine proteases enhanced immunogenicity.  To determine whether there was an active suppression of the murine humoral response, ECP was co-administered with highly immunogenic oyster hemolymph.  ECP substantively suppressed the specific antibody response to hemolymph and decreased recognition of the number of epitopes.  Analysis of media constituents revealed that the known immunomodulatory surfactant, Pluronic F-68 (PF68), used in the defined lipid concentrate supplement, elicited significant immunosuppression.  Although isolated ECP antigens were immunosuppressive, their separation from the PF68 enabled production of nine monoclonal antibodies (MAbs), which recognized many of the ECPs.  These MAbs fell into three groups.  Antibodies in the first group recognized an approximate 31-kDa protein and several apparent degradation products.  By immunofluorescence assay, these antibodies bound either internal vesicles or a surface protein with a patchy distribution.  The second group of MAbs bound a number of proteins and brightly stains the cell surface.  These antibodies have been used to detect P. marinus in formalin-fixed, paraffin-embedded oyster tissue sections by immunohistochemistry.  Immunogold electron microscopy demonstrated that this group specifically bound the cell wall as well as one type of intracellular vesicle.  The third group apparently bound a protein and its approximate 100 and 20 kDa fragments.  Immunofluorescence demonstrates binding similar to that of the first group.  Several combinations of the antibodies are able to detect ECP in a capture ELISA format.  Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page