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Purification and identification of a Dicrocoelium dendriticum specific antigen

AutorRevilla-Nuín, Beatriz; Manga-González, M. Yolanda ; Pozo Carnero, María Paz del; González Lanza, Camino
Fecha de publicaciónjul-2004
EditorEuropean Federation of Parasitologists
CitaciónIX European Multicolloquium of Parasitology (EMOP IX); 509 (P) (2004)
ResumenThe immunological diagnosis and the immunoprophylaxis of dicrocoeliosis require the study of antigens of Dicrocoelium dendriticum, the parasite producing that parasitosis on the Iberian Peninsula. In a previous research we detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels. It also showed strong immunoreactivity by westem-blot against ovine sera experimentally infected with D. dendriticum. The aim of the current study is the purification and identification of that protein. For this, in the first place, we carried out a fractioned precipitation with ammonium sulphate for parasite somatic extracts and, for the excretion-secretion products, a concentration by ultrafiltration. In both cases the samples were subsequently analyzed by chromatographic techniques of gel filtration (Sephacryl S-300), ion exchange (Hitrap DEAE-Sepharose-FF) and affinity (Dye ligand reagent Green 19). The search for and checking of 130 kDa protein among the different chromatographic fractions was done by SDS-PGAE (12% gels) and western-blot. Finally, the N-amino terminal acid sequencing of the purified protein was determined by automated Edman degradation. To verify the serodiagnostic value of the 130 kDa protein indirect ELISA assays were done. The protein purified by DEAE-Sepharose and later eluted from 8% gels was used as the antigen. The best chromatographical results obtained were with Hitrap DEAE-Sepharose-FF-column. The elutions conditions of that protein were similar to that from somatic antigen (pH 7.2, 0.1 M NaCI, in 29 to 34 ml fractions) and from excretion-secretion antigens (pH 8.0, 0.1 M NaC1, in 29 to 35 ml fractions) The amino terminal sequence analysis confirmed the similarity between both fractions and revealed homology with a D. dendriticum hemoglobin. The results obtained by ELISA showed that the 130 kDa protein discriminates positive and negative D. dendriticum experimental sera from ovine and rabbits and can therefore be used for the serological diagnosis of dicrocoeliosis.
Descripción1 page.-- Poster presentado al IX European Multicolloquium of Parasitology (Valencia, España, 18-23 July, 2004)
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