Separação imunomagnética de cistos teciduais e esporocistos de toxoplasma gondii pelo anticorpo monoclonal K8/15-15 e caracterização de sua proteína alvo por Western Blot e Imunoblot

Abstract

Toxoplasma gondii is an obligatory intracellular parasite that infects about one third of the human population. Horizontal transmission of T. gondii occurs more often by ingestion of tissue cysts containing bradyzoites and/or ingestion of sporulated oocysts. The current study aimed to develop a protocol of immunomagnetic separation (IMS) directed to T. gondii tissue cysts and soporocysts, and to isolate the protein recognized by the monoclonal antibody (mAb) K8/15-15. This was the first described mAb that is able to simultaneously recognize proteins in tissue cyst and sporocyst walls of the parasite. Using IMS, more than 2,000 in vitro produced tissue cysts were isolated from a single culture flask of 25 cm2 . Moreover, the ratio of tissue cyst/host cell was increased six times by IMS. In vivo generated cysts were successfully separated from murine cells, with no contamination by cell debris or mouse cells. Sporocysts were also separated by IMS, showing that this method may be employed for proteomic studies involving tissue cyst and sporocyst walls of T. gondii. Using IMS, a great amount of tissue cysts was concentrated from cell culture, and after immunoprecipitation, the target antigen was separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE). The antigen was processed by Western blot, and bands of 272 and 381 kDa were labeled by IMUNOBLOT. The same bands were observed in a separate SDS-PAGE stained by Comassie blue. The results obtained here will favor the conduction of further experiments to fully characterize the target protein of the mAb K8/15-15, in particular, by sequencing the isolated protein by mass spectrometry. The purified protein may be tested in serological studies, as well as in vaccines to prevent toxoplasmosis.