Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera – A phase II study

Abstract 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.