Use of Recombinant Antigens for the Accurate Immunodiagnosis of Chagas' Disease

Abstract

We tested two Trypanosoma cruzi recombinant antigens in a diagnostic test for Chagas' disease. These antigens were a cytoplasmic repetitive antigen (CRA) and a flagellar repetitive antigen (FRA). The results indicate that the recombinant antigens give better results when used in combination than when used separately, and that the removal of the beta-galactosidase portion of the recombinant fusion proteins increases the specificity of the diagnostic test for Chagas' disease. In addition, a direct enzyme-linked immunosorbent assay (ELISA), which involves the use of peroxidase-labeled antigens to detect the immune-complexes, was developed and compared with a conventional ELISA. The results indicate that the recombinant (CRA+FRA) ELISA is better than the conventional ELISA in the diagnosis of Chagas' disease, providing 100% specificity and sensitivity in all sera tested to date. The recombinant ELISA was compared with conventional serologic tests (hemagglutination and immunofluorescence) for Chagas' disease diagnosis, and the results show that the recombinant ELISA does not give rise to false-positive results that are observed with the two other tests. The use of the recombinant ELISA should be useful in the prevention of transmission of Chagas' disease by blood transfusions.

Diagnosis of Chagas disease: what has been achieved? What remains to be done with regard to diagnosis and follow up studies?

“…Several research groups have therefore used recombinant/synthetic and biochemically purified antigens (Affranchino et al 1989, Goldenberg et al 1991, Krieger et al 1992, Umezawa et al 1999, Franco da Silveira et al 2001. In order to be useful, these antigens must meet several criteria: (i) they should be present in T. cruzi isolates from different endemic areas, and absent from other infectious disease agents; (ii) they should be highly immunogenic in populations with different genetic backgrounds, irrespective of the clinical phase of Chagas disease and (iii) they should be stable and easily amenable to qualitycontrol tests, to guarantee reproducibility (Zingales et al 1990, Stolf 1992).…”

“…Even though the majority of these are tested and used in research with satisfactory results, they have not been included in kits for technical and economic reasons (Franco da Silveira et al 2001). Two recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (Lafaille et al 1989), have been deemed useful for diagnostic purposes, according to a multicentre study carried out by the WHO (Almeida et al 1990, Moncayo & Luquetti 1990, Paranhos et al 1990, Krieger et al 1992, Carvalho et al 1993. A kit for diagnosis of chronic Chagas disease called EIE-Recombinante-ChagasBiomanguinhos ® (Rec-ELISA), which uses these antigens in combination (CRA + FRA), has been developed and produced by Biomanguinhos, at the Oswaldo Cruz Foundation (Fiocruz), in Rio de Janeiro, Brazil.…”

Expression of co-stimulatory molecules CD80 and CD86 is altered in CD14 + HLA-DR + monocytes from patients with Chagas disease following induction by Trypanosoma cruzi recombinant antigens

“…This may prevent the detection of a specific immune response as these preparations include a complex mixture of antigens. Therefore, the use of defined parasite reagents such as recombinant antigens becomes an important requirement for the identification of molecules critical for parasite-host interactions and in studies of the immunopathology of CD (6) (7) . Cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (6) (7) have been used by our group to study the development of immunological markers for the prognosis of chronic clinical forms of CD.…”

“…CRA and FRA were obtained as described by Krigger et al (7) , and were prepared at the Department of Diagnostic Reagents of Bio-Manguinhos before being sent to the Immunoparasitology Laboratory at Centro de Pesquisa Aggeu Magalhães/Fundação Oswaldo Cruz (CPqAM/FIOCRUZ). They were further evaluated for any contamination with bacterial proteins and carbohydrates, as described by Pereira et al (8) Heparinized peripheral blood was cultured for about 24h and diluted 1:1 in 14mL polypropylene tubes (BD Biosciences, San Jose, CA, USA) containing RPMI 1640 medium supplemented with L-glutamine, 1% antibiotics (stock solution of 10,000U penicillin and 10,000U streptomycin; Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (Sigma-Aldrich).…”

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

“…CRA (cytoplasmic repetitive antigen) and FRA (flagellar repetitive antigen) recombinant antigens (RecAg) of T. cruzi have been used, successfully in serodiagnosis of Chagas disease (Krieger et al 1992, Gomes et al 2001. FRA is located in the flagellum of the epimastigote and trypomastigote forms of T. cruzi and displays a 68-amino acid repeat, while CRA is distributed throughout the cytoplasm of epimastigote and amastigote forms and has a 14-amino acid repeat (Lafaille et al 1989 The response of chagasic patients to these antigens (Krieger et al 1992, Gomes et al 2001) led us to investigate their immunologial response in experimental animals.…”