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The chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4 perform well in indirect formats, and a proof-of-concept phase I double-antigen sandwich ELISA (DAgS-ELISA) achieved high specificity but limited sensitivity. We re-optimized and validated the IBMP-DAgS-ELISA in a phase II study.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      The four antigens were conjugated to horseradish peroxidase and re-optimized by checkerboard titration. Diagnostic performance was assessed against a latent class analysis reference standard using 403\n                      <jats:italic>T. cruzi<\/jats:italic>\n                      -positive and 399\n                      <jats:italic>T. cruzi<\/jats:italic>\n                      -negative sera from the Central Public Health Laboratory of Bahia, applying a single pre-specified reactivity-index cut-off (RI\u2009\u2265\u20091.0). Cross-reactivity was evaluated in 206 sera from individuals with other infectious diseases, and intra-plate repeatability in 28 positive and 28 negative sera tested in triplicate.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      IBMP-8.4 showed the highest values for most performance measures (area under the curve 99.8%, sensitivity 99.8%, specificity 98.0%, accuracy 98.9%, diagnostic odds ratio 19,648, Cohen\u2019s \u03ba 0.98), and IBMP-8.3 the highest specificity (99.0%). The parallel combination IBMP-8.3\/IBMP-8.4 reached 100% sensitivity and 97.0% specificity. Cross-reactivity was low overall and absent for IBMP-8.4 (0\/206), including against\n                      <jats:italic>Leishmania<\/jats:italic>\n                      and\n                      <jats:italic>Schistosoma<\/jats:italic>\n                      spp. Across triplicate testing, sensitivity (92.9\u2013100%) and area under the curve (94.3\u2013100%) were stable for all antigens, whereas specificity varied more widely (78.6\u2013100%) in the repeatability subset.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The re-optimized IBMP-DAgS-ELISA matches the indirect-ELISA benchmark of the same antigens while detecting total antibodies independently of immunoglobulin class or host species. IBMP-8.4, alone or combined with IBMP-8.3, is an accurate option for chronic CD serology and reinstates a double-antigen sandwich platform on a fully defined chimeric-antigen basis.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s10096-026-05687-y","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T23:59:50Z","timestamp":1790639990000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera \u2013 A phase II study"],"prefix":"10.1007","author":[{"given":"Nat\u00e1lia Erdens Maron","family":"Freitas","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felipe Silva Santos","family":"Jesus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Randrin Queiroz Viana","family":"Ferreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edimilson Domingos","family":"Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keila Gisele Azevedo Figueiredo","family":"Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Am\u00e9lia Virgens","family":"Lima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristiane Oliveira","family":"Mota","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u00c2ngelo Ant\u00f4nio Oliveira","family":"Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paola Alejandra Fiorani","family":"Celedon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nilson Ivo Tonin","family":"Zanchin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3944-0818","authenticated-orcid":false,"given":"Fred Luciano Neves","family":"Santos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"5687_CR1","unstructured":"Pan American Health Organization. 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