European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2026
Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera - A phase II study.
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Freitas Natália Erdens Maron, Jesus Felipe Silva Santos, Ferreira Randrin Queiroz Viana, Silva Edimilson Domingos, Santos Keila Gisele Azevedo Figueiredo, Lima Maria Amélia Virgens, Mota Cristiane Oliveira, Silva Ângelo Antônio Oliveira, Celedon Paola Alejandra Fiorani, Zanchin Nilson Ivo Tonin, Santos Fred Luciano Neves
Аннотация
PURPOSE: Laboratory confirmation of chronic Chagas disease (CD) relies on serology, yet conventional indirect ELISAs depend on species- and class-specific secondary antibodies and remain prone to cross-reactivity. 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.
METHODS: 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 T. cruzi-positive and 399 T. cruzi-negative sera from the Central Public Health Laboratory of Bahia, applying a single pre-specified reactivity-index cut-off (RI ≥ 1.0). 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.
RESULTS: 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's κ 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 Leishmania and Schistosoma spp. Across triplicate testing, sensitivity (92.9-100%) and area under the curve (94.3-100%) were stable for all antigens, whereas specificity varied more widely (78.6-100%) in the repeatability subset.
CONCLUSION: 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.
METHODS: 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 T. cruzi-positive and 399 T. cruzi-negative sera from the Central Public Health Laboratory of Bahia, applying a single pre-specified reactivity-index cut-off (RI ≥ 1.0). 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.
RESULTS: 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's κ 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 Leishmania and Schistosoma spp. Across triplicate testing, sensitivity (92.9-100%) and area under the curve (94.3-100%) were stable for all antigens, whereas specificity varied more widely (78.6-100%) in the repeatability subset.
CONCLUSION: 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.