RESUMO
Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982).
Assuntos
Antígenos CD/genética , Antígenos de Protozoários , Clonagem Molecular , Proteínas Inativadoras do Complemento/genética , Glicoproteínas de Membrana/genética , Proteínas de Protozoários , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Sequência de Bases , Antígenos CD55 , DNA/genética , DNA/isolamento & purificação , Humanos , Soros Imunes/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Coelhos , Trypanosoma cruzi/genéticaRESUMO
In this study we describe a simple and rapid method that uses sulfo-N-hydroxy-succinimidobiotin (sulfo-NHS-biotin) to label Trypanosoma cruzi surface proteins stably without significant loss of biological function. Efficient labelling can be obtained with as little as a 5 minute incubation of parasites in an appropriate concentration of sulfo-NHS-biotin at 4 degrees C. After labelling under these conditions, biotinylated parasites exhibited levels of motility, viability, and in vivo infectivity comparable to those seen with unlabelled control parasites. Moreover, the biological activity of T-DAF, a complement regulatory protein found on the parasite surface, was unaffected when biotinylated under these conditions. Biotinylated surface proteins can be easily detected in a variety of non-radioactive assays employing conjugated streptavidin as a developer. Compared to alternative techniques of surface labelling described in the literature, this method offers better preservation of biological function as well as greater ease of use and safety.