Purified excreted-secreted antigens from Trypanosoma cruzi trypomastigotes as tools for diagnosis of Chagas' disease

Abstract

There is currently no "gold standard" test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (n = 195), healthy controls (n = 400), and patients with other parasitic diseases (n = 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA(IA) proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA(IA) proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania. These data suggest the need for field validation studies of TESA- and TESA(IA)-based assays in regions where Chagas' disease is endemic.

FIG. 1.

SDS-PAGE separation of the immunopurified proteins from total concentrated TESA and TESA_IA visualized by silver staining. (A) Tulahuen strain (total concentrated TESA is in the leftmost lane; fractions 7 to 10; the peak is in fraction 9); (B) Brazil strain (fractions 22 to 28; the peak is in fraction 23).

FIG. 2.

Western blot analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated total Tulahuen strain TESA (A) and TESA_IA (B) preparations of T. cruzi. The dot was probed with a pool of sera from patients with Chagas' disease.

FIG. 3.

Western blot analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins purified by affinity chromatography from total TESA of T. cruzi (Tulahuen strain). Lanes: 1, Chagas disease-positive sera; 2 and 3, sera from healthy patients; 4 and 5, Leishmania-positive sera (lane 4, reactive with the 60-kDa band; lane 5, nonreactive with the 60-kDa band). Arrow, 60-kDa band that is cross-reactive.