Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease
An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease , healthy controls , and patients with other parasitic diseases was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220？kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease. 1. Introduction Chagas disease, discovered by Dr. Carlos Chagas in 1909, is caused by the protozoan parasite, Trypanosoma cruzi. This parasite can be transmitted by triatomine vectors, blood transfusions, laboratory accidents, organ transplantation, or through vertical transmission. The disease is endemic in 18 Latin American countries. In 2008, the World Health Organization (WHO) estimates that ~8 million people were infected resulting in ~11？000 deaths per year [1]. Despite decades of effort, there is still no gold standard test for the diagnosis of Chagas disease and several international organizations have recently emphasized the need for improved serodiagnostic tests [1]. To this end, many companies and research groups have developed a range of parasitologic, serologic, and nucleic acid-based assays. The most common methods include the indirect immunofluorescence assay (IFA), indirect haemagglutination assay (IHA), and the enzyme-linked immunosorbent assay (ELISA) [2]. The target antigens in these latter assays have included whole fixed parasites [3], 67 and 90-kDa lectin purified proteins [4, 5], synthetic peptides [6], recombinant proteins [7–14], and trypomastigote excreted/secreted antigen (TESA). Of these antigens, TESA proteins have shown particular promise in different assay formats (ELISA-TESA, Western blot) and in several different laboratories and geographic settings [15–19]. Unfortunately, the production of TESA antigen requires adequate cell culture facilities and other sophisticated infrastructure that are not always available to laboratories in resource-poor settings. Thus, in
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