Role of the rhoptry proteins of sporozoites during hepatocyte invasion by Plasmodium

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All invasive forms of Apicomplexa possess a complex of organelles located at the anterior end of the parasite. The organelles comprise rhoptries, dense granules and micronemes. The rhoptries are pear shaped, electron-dense, membrane bound organdies and are considered to have roles in host cell attachment, invasion and formation of parasitophorous vacuole membrane (PVM). Membranous materials are secreted from the rhoptries during the invasion. These materials are thought to be incorporated into the expanding PVM.Several proteins of varying molecular weight have been identified in P.falciparum merozoite rhoptries, but apical organelles of sporozoite have not been investigated. To analyze the rhoptry proteins of sporozoites, we try to produce monoclonal antibodies against sporozoite rhoptry proteins of rodent malaria. P.berghei sporozoites were enriched and were disrupted by nitrogen decompression method in order to release the organelles in a mild condition. Organelles were then, purified by sucrose density gradient centrifugation. Subcellular fractions were observed by the electron microscopy. We immunized BALB/c mice with these fractions or disrupted native sporozoites. We are trying to clone the hybridoma which produce the monoclonal antibody against P.berghei sporozoite apical organelles by using ELISA or IFA test.