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Clin. Vaccine Immunol. doi:10.1128/CVI.00401-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Immunoblot Assay Using Recombinant Antigens As a Supplemental Test to Confirm Antibodies to Trypanosoma cruzi

Emerging Pathogens and Infectious Diseases R&D, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois, Department of Internal Medicine, Infectious Diseases, University of Iowa, Iowa City, Iowa and Transmissible Diseases Department, American Red Cross, Rockville, Maryland

^* To whom correspondence should be addressed. Email: dinesh.shah{at}abbott.com.

	Abstract

The diagnosis of chronic Chagas' disease is generally made by detecting antibodies to Trypanosoma cruzi. Most conventional serological tests are based on lysates of whole parasites or semi-purified antigen fractions from T. cruzi epimastigotes grown in culture. The occurrence of inconclusive and false-positive results has been a persistent problem with the conventional assays, and there is no universally accepted gold standard for confirmation of positive test results. We describe here an immunoblot assay for detecting antibodies to T. cruzi in which four chimeric recombinant antigens (rAgs), designated FP3, FP6, FP10, and TcF, are used as target antigens. These rAgs are each composed of several antigenically distinct regions and include repetitive as well as non-repetitive sequences. Each rAg is coated as a discrete line on a nitrocellulose strip. Assay sensitivity was assessed by testing 345 specimens known to be positive for antibodies to T. cruzi. All 345 of these samples showed two to four reactive test bands, in addition to the three on-board control bands that are on each strip. Assay specificity was determined by testing 500 specimens from random U.S. blood donors, all of which were negative. Based on the results obtained in this study, we propose the following scheme for interpretation of test results: a) no bands or a single test band - negative; b) two or more test bands with at least one band having intensity of 1+ or higher - positive; and c) multiple faint test bands (+/-) - indeterminate. Based on this scheme, the prototype immunoblot assay showed a sensitivity of 100% (n = 345) and a specificity of 100% (n = 500). Additionally, all 269 potentially cross-reacting and T. cruzi antibody negative specimens tested negative in our immunoblot assay. The rAg-based immunoblot assay has potential as a supplemental test for confirming the presence of antibodies to T. cruzi in blood specimens and for identifying false positive results obtained with other assays.