Detection method for carrying Hepatitis B surface antigen exosomes by a chemiluminescence immunoassay (CLIA)

Conventional detection of Hepatitis B surface antigen (HBsAg) cannot differentiate exosome-associated antigen from its free form, which may hold distinct biological significance. Here, we developed a specific chemiluminescence immunoassay (CLIA) to detect HBsAg-positive, CD63-positive particles (putative HBsAg-carrying exosomes).

The assay employs magnetic beads coated with an anti-HBsAg antibody for exosome capture, followed by detection using an acridinium ester-labeled anti-CD63 antibody. All performance evaluations were conducted in normal human serum as the biological matrix.

Analytical validation established cutoff value of 4,421 RLU from 80 blank replicates (per CLSI EP17-A2). The method demonstrated complete analytical agreement with the engineered reference samples tested (n = 15 negative; n = 5 positive). The limit of detection was 7.8 × 10⁸ particles/mL, corresponding to a sample dilution factor of 128. Intra- and inter-assay precision yielded coefficients of variation below 15% across the measuring range.

This CLIA provides an analytically validated qualitative screening tool for research use (classifying samples as positive or negative based on the established cutoff of 4,421 RLU) for the in vitro detection of HBsAg-positive, CD63-positive particles, facilitating future research into the role of exosomal cargo in Hepatitis B virus pathogenesis.