This supplementary material provides detailed information on the methods and findings of a study investigating the role of antibody-dependent enhancement (ADE) in severe dengue disease. The study utilized the Nicaraguan Pediatric Dengue Cohort Study (PDCS), which has been ongoing since 2004, to measure anti-dengue virus binding antibody (DENV-Ab) titers using an Inhibition ELISA (iELISA). The iELISA was developed to measure functional antibodies that block DENV-specific peroxidase-conjugated IgG binding to DENV antigens. The study included 6,684 participants and measured DENV-Ab titers annually over a 12-year period. Key findings include: 1. **Quality Control and Reproducibility**: The iELISA was validated through proficiency panels and quality control measures, ensuring consistent and reliable results. 2. **Comparison with Other Assays**: The iELISA was compared to the hemagglutination inhibition assay (HI) and a flow-cytometry-based neutralization assay, showing strong correlations and sensitivity. 3. **DENV-Ab Titer Analysis**: The relationship between iELISA titers and neutralizing antibody titers was analyzed, revealing a strong correlation, especially for heterotypic DENV types. 4. **Severe vs. Non-severe Dengue**: Cox proportional hazards models were used to estimate the hazard ratios for severe dengue (DHF/DSS) and symptomatic dengue by pre-existing DENV-Ab titer, adjusting for covariates such as sex, epidemic season, age, and number of previous infections. 5. **Antibody Decay**: The rate of DENV-Ab decay over time was estimated, showing a half-life of approximately 4 years. 6. **Nested Case-Control Analysis**: A nested case-control design was used to evaluate the association between pre-existing DENV-Ab titers and the severity of secondary dengue cases. 7. **Transmission Implications**: The potential impact of ADE on DENV transmission was discussed, highlighting the need for a better understanding of viremia dynamics and transmission probability. The study provides robust evidence that pre-existing DENV-Ab titers can predict the severity of dengue disease, with higher titers associated with a reduced risk of severe outcomes.This supplementary material provides detailed information on the methods and findings of a study investigating the role of antibody-dependent enhancement (ADE) in severe dengue disease. The study utilized the Nicaraguan Pediatric Dengue Cohort Study (PDCS), which has been ongoing since 2004, to measure anti-dengue virus binding antibody (DENV-Ab) titers using an Inhibition ELISA (iELISA). The iELISA was developed to measure functional antibodies that block DENV-specific peroxidase-conjugated IgG binding to DENV antigens. The study included 6,684 participants and measured DENV-Ab titers annually over a 12-year period. Key findings include: 1. **Quality Control and Reproducibility**: The iELISA was validated through proficiency panels and quality control measures, ensuring consistent and reliable results. 2. **Comparison with Other Assays**: The iELISA was compared to the hemagglutination inhibition assay (HI) and a flow-cytometry-based neutralization assay, showing strong correlations and sensitivity. 3. **DENV-Ab Titer Analysis**: The relationship between iELISA titers and neutralizing antibody titers was analyzed, revealing a strong correlation, especially for heterotypic DENV types. 4. **Severe vs. Non-severe Dengue**: Cox proportional hazards models were used to estimate the hazard ratios for severe dengue (DHF/DSS) and symptomatic dengue by pre-existing DENV-Ab titer, adjusting for covariates such as sex, epidemic season, age, and number of previous infections. 5. **Antibody Decay**: The rate of DENV-Ab decay over time was estimated, showing a half-life of approximately 4 years. 6. **Nested Case-Control Analysis**: A nested case-control design was used to evaluate the association between pre-existing DENV-Ab titers and the severity of secondary dengue cases. 7. **Transmission Implications**: The potential impact of ADE on DENV transmission was discussed, highlighting the need for a better understanding of viremia dynamics and transmission probability. The study provides robust evidence that pre-existing DENV-Ab titers can predict the severity of dengue disease, with higher titers associated with a reduced risk of severe outcomes.