Immunoagglutination Assay for the Diagnosis of Trypanosoma cruzi Infection

GARCIA VALERIA; GONZALEZ VERONICA; VEGA JORGE; MARCIPAR IVÁN; GUGLIOTTA LUIS

Workshop; First Workshop on Advances in Colloidal Materials; 2011

Immunological methods are nowadays the elective procedure to diagnose Chagas disease. This illness caused by infection with the parasite Trypanosoma cruzi, affects between 16 and 18 million people only in Latin America, with a further 100 million considered at risk. Chagasic infection is mostly diagnosed when specific antibodies (Abs) against T. cruzi antigens (Ags) are detected in patients blood. Total homogenate of the parasite at the epimastigote stage provides Ags for serological tests, since it was proved to render the appropriate sensitivity to detect low Ab levels. However, when using this complex mixture of variable, largely undefined Ags, not only specificity problems appear but also difficulties to standardize the methodology. The current trend is using recombinant proteins as sensitizing elements, since large amounts of them can be obtained in a highly purified form, and additionally, they can be synthesized from DNA sequences engineered to encode peptide fragments where the specific regions responsible of cross-reactivity have been excised. Bearing in mind the sensitivity loss when using single recombinant peptides, several authors have suggested that a peptide mixture would reach a sensitivity equal to the sum of that of the individual Ags. Alternatively, the use of multiepitope proteins expressing several unrelated antigenic determinants has also been proposed to enhance sensitivity. The latex agglutination test which involves in vitro aggregation of latex particles coated with Ag (or Ab), called latex-Ag (or Ab) conjugate, in the presence of specific Ab (or Ag) is of great interest especially in biomedical applications. This test is claimed to be the quickest and easiest method. The agglutination reaction produced during the immunoassay can be detected by either visual method or instrumental-based techniques. The final aim is the production of an immunoagglutination kit for diagnosing the Chagas disease. The work can be divided into 5 stages: I) the controlled synthesis of latex particles; II) the surface and size characterization of the latex particles; III) the expression of recombinant proteins of T. cruzi; IV) the sensitization of particles with the antigenic protein; and V) the application of the latex-protein complex in immunoagglutination assays.