Development of a hypersensitive digital ELISA system for hepatitis B core antigen

Area of Science:

• Hepatology
• Virology
• Biotechnology

Background:

• Chronic hepatitis B (CHB) is a significant global health issue caused by the hepatitis B virus (HBV).
• Current treatments, nucleos(t)ide analogs (NUCs), suppress HBV replication but cannot eliminate the viral cccDNA, necessitating lifelong therapy due to relapse risk.
• Accurate monitoring of cccDNA activity is crucial for NUC discontinuation, but existing HBV biomarker detection methods lack sensitivity, especially at low concentrations.

Purpose of the Study:

• To develop a highly sensitive and reliable biomarker assay for monitoring CHB, correlating with cccDNA levels.
• To establish a digital enzyme-linked immunosorbent assay (ELISA) system for detecting hepatitis B core antigen (HBcAg).

Main Methods:

• Development of a digital ELISA system utilizing a pair of monoclonal antibodies specific for HBcAg.
• Assessment of the system's sensitivity and detection limit for HBcAg.
• Evaluation of the influence of plasma and serum matrices on assay sensitivity and specificity.

Main Results:

• The digital ELISA system achieved a detection limit of 2.9 pg/mL (58 aM) for HBcAg, representing a ~1000-fold increase in sensitivity compared to conventional ELISA.
• Assay sensitivity remained unaffected by plasma and serum matrices at concentrations of 1% or less.
• The digital ELISA demonstrated specific reactivity with HBcAg, even in the presence of biological matrices.

Conclusions:

• The developed highly sensitive digital ELISA system for HBcAg detection is a promising tool for CHB management.
• This assay could enable more accurate monitoring of therapeutic strategies and potentially guide NUC discontinuation.
• Further validation is warranted to establish HBcAg digital ELISA as a reliable biomarker for CHB treatment evaluation.