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Comparative Study
. 2009 Jun;16(6):899-905.
doi: 10.1128/CVI.00005-09. Epub 2009 Apr 1.

Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis

Cecilia Camussone  1 , Verónica Gonzalez, María S Belluzo, Nazarena Pujato, María E Ribone, Claudia M Lagier, Iván S Marcipar
Affiliations
Comparative Study

Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis

Cecilia Camussone et al. Clin Vaccine Immunol. 2009 Jun.

Abstract

The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic representation of the construction and methods used to obtain the plasmids encoding the better-performing recombinant peptides selected, RP1, RP2, and RP5, and the chimeric proteins CP1 and CP2. (B) Amino acid sequences of the multiepitope chimeric proteins CP1 and CP2.
FIG. 2.
FIG. 2.
Relative OD distribution obtained for a panel of 32 Chagas' disease-positive serum specimens, using all the Ags studied. Horizontal lines show the discrimination efficiency values (the mean OD values of the positive samples tested divided by the cutoff value). (A) TPH and the isolated peptides RP1, RP2, and RP5. (B) RP1+RP2 mixture and RP1+RP2+RP5 mixture. (C) CP1 (fused RP1-RP2) and CP2 (fused RP1-RP2-RP5).
FIG. 3.
FIG. 3.
Relative OD distribution obtained using CP2 for a panel of 109 Chagas' disease-positive (Pos) and 132 Chagas' disease-negative (Neg) serum samples, together with 15 serum samples from individuals infected with Leishmania spp. (Leish). Horizontal lines show the discrimination efficiency values (mean OD values of the positive samples tested divided by the cutoff value).
FIG. 4.
FIG. 4.
Illustration of the well-sensitizing step, which is followed by the Ag-Ab reaction for the RP1+RP2+RP5 peptide mixture and the CP2 chimeric protein bearing the fused RP1-RP2-RP5 peptides.
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