Camussone Cecilia, Gonzalez Verónica, Belluzo María S, Pujato Nazarena, Ribone María E, Lagier Claudia M, Marcipar Iván S
Laboratorio de Tecnología Biológica, Universidad Nacional del Litoral, Santa Fe, Argentina.
Clin Vaccine Immunol. 2009 Jun;16(6):899-905. doi: 10.1128/CVI.00005-09. Epub 2009 Apr 1.
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.
这项工作的目的是确定在免疫化学装置上展示抗原（Ag）的最佳策略，以提高检测的选择性和灵敏度。我们对五种克氏锥虫抗原重组肽进行了比较评估，选择了三种敏感性更高的肽，构建了携带这些选定抗原的嵌合体，并通过酶联免疫吸附测定法系统地比较了这些肽组合与线性融合所有这些肽的多表位构建体的性能。所比较的性能较好的抗原包括与先前描述的克氏锥虫鞭毛重复抗原（此处命名为RP1）、急性期释放抗原（RP2）、B13（RP5）同源的肽，以及分别携带RP1-RP2和RP1-RP2-RP5重复序列的嵌合重组蛋白CP1和CP2。通过公式+OD/临界值（其中+OD是测试的阳性血清样本的平均光密度值）评估这些抗原的诊断性能，以比较其单独、混合或作为肽融合嵌合体时彼此之间以及与总寄生虫匀浆（TPH）的鉴别效率。CP1和CP2获得的鉴别效率值分别比其单个抗原混合物高25%和52%。与TPH相比，CP2是唯一在恰加斯病阳性和阴性样本之间显示出更高鉴别效率的抗原。这项研究突出了使用杂交、单分子、嵌合抗原而非肽混合物进行免疫化学分析的便利性。当用141个恰加斯病阳性血清样本面板进行评估时，CP2的初步测试灵敏度为98.6%，当用包含15个来自感染利什曼原虫属个体的样本的164个恰加斯病阴性血清样本面板进行评估时，特异性为99.4%。
