Enhanced HBcAg Immunoassay Enables Detection of Clinically Relevant Low-Level HBV Viremia for Treatment Eligibility

Accurate staging of chronic hepatitis B is essential for guiding treatment decisions and predicting clinical outcomes. Current treatment guidelines rely on HBV DNA quantification, with therapeutic thresholds set at ≥2,000 IU/mL or ≥20,000 IU/mL depending on liver enzyme levels, HBeAg status, and evidence of fibrosis or cirrhosis. Despite these guidelines, access to molecular diagnostics for HBV DNA measurement remains limited in many low- and middle-income countries (LMICs) where most infections occur, creating barriers to timely and effective care. Importantly, non-phosphorylated HBcAg, located in HBV DNA-containing particles, has emerged as surrogate biomarker for HBV DNA. However, more sensitive HBcAg immunoassays could enable detection at clinically relevant thresholds and improve disease management. We modified a first-generation (v1) HBcAg-specific assay by incorporating a new formulation and monoclonal antibodies, and implemented the updated assay on a digital ELISA platform (v2). HBcAg levels were analyzed in longitudinal specimens from acute HBV patients and single timepoint specimens from treatment-naïve blood donors. The enhanced assay (v2) achieved a limit of quantification of 30 fg/mL, corresponding to ~1,000 HBV DNA IU/mL. Comparative analysis demonstrated a reduction in the detection threshold from 4.9×10 5 to 1,000 HBV DNA IU/mL, aligning with treatment eligibility criteria. Clinical sensitivity improved on average 225-fold in patient specimens. The HBcAg v2 assay enables detection of low viral titers consistent with treatment guidelines and correlates directly with HBV DNA levels. Its ability to specifically detect low-abundance HBcAg enhances disease staging and may support therapeutic management, particularly in resource limiting settings. • HBcAg v2 assay improves analytical sensitivity 460-fold over v1 • Detects HBV at ~1,000 DNA IU/mL, aligning with treatment thresholds • Strong correlation with HBV DNA (R² = 0.98) in clinical samples • No cross-reactivity; clinical sensitivity improved 225-fold • Cost-effective alternative to determine treatment eligibility in resource-limited settings