Monoclonal antibody analysis of Perkinsus marinus extracellular products

• Published in: International journal for parasitology 2005-02, Vol.35 (2), p.171-184
• Main Authors: Earnhart, Christopher G., Gauthier, David T., Vogelbein, Wolfgang K., Kaattari, Stephen L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Animal aquaculture; Animal productions; animal tissue extracts; Animals; Antibodies, Monoclonal - analysis; antigen detection; antigen-antibody reactions; Biological and medical sciences; Crassostrea virginica; Enzyme-Linked Immunosorbent Assay - methods; Eukaryota - immunology; Eukaryota - ultrastructure; excretory-secretory products; Extracellular products; Extracellular Space - immunology; Fluorescent Antibody Technique - methods; Fundamental and applied biological sciences. Psychology; immunochemistry; Immunohistochemistry - methods; Invertebrate aquaculture; Life cycle. Host-agent relationship. Pathogenesis; Marine; Microscopy, Electron; Mollusca; Monoclonal antibodies; Nuclear Proteins - analysis; Ostreidae - parasitology; oyster homogenates; oysters; Perkinsus marinus; protein synthesis; Protozoa; Protozoan Proteins - analysis; Protozoan Proteins - immunology; purification; quantitative analysis; Retinoblastoma-Like Protein p107; Virulence; Virulence factors
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2004.10.025

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.