Supporting Method
Quantitative HBeAg assay
The development of the quantitative HBeAg assay was based on the method of
Perrillo et al (14). Serum HBeAg was measured by EIA (ARCHITECT platform; Abbott
Laboratories). This commercial HBeAg kit is not marketed as a quantitative assay but
produces a signal which is linear within a restricted range. The HBeAg reference
preparation was obtained from the Paul Ehrlich (PE) Institute (Paul-Ehrlich Institute,
Langen, Germany). The preparation has a defined HBeAg activity of 100 PE IU/mL. An
in-house working HBeAg standard was then prepared from a pool of high-titre HBeAg
positive specimens received for routine diagnostic testing, and calibrated against the PE
reference preparation. The standard was subsequently determined to have an HBeAg titre
of 2900 PE IU/mL, and was then used to generate a calibration curve within the linear
range of the assay. Dilutions of the in-house working standard (diluted twofold, from 1 in
2 to 1 in 4,096, or 1450 to 0.7 PE IU/mL) were prepared using the ARCHITECT HBsAg
Manual Diluent (Abbott Diagnostics, Sligo, Ireland), which contains recalcified human
plasma that is non-reactive for HBsAg, HIV-1 RNA or HIV-1 Ag , anti-HIV-1/HIV-2,
anti-HCV, and anti-HBs. The standards were aliquoted and stored at -20C.
The performance characteristics of the assay for use as a quantitative tool were
evaluated. The in-house HBeAg working standard was used to generate a calibration
curve within the linear range of the assay. The linear range was approximately 0.5 – 100
PE IU/mL. For determination of the inter-assay variability, 8 dilutions of the standard (1
in 2 – 1 in 4096) were tested in four separate runs. No significant deviation from linearity
was present for any of the following dilutions: 1 in 16, 1 in 32, 1 in 64, 1 in 128, 1 in 256,
1 in 512, 1 in 1024 and 1 in 2048 (data not shown). Intra-assay variation was tested using
duplicate samples of the reference preparation at 1 in 64 and 1 in 2048 dilutions, in four
separate assay runs. The percentage coefficient of variation ranged from 0.68 – 7.6%,
with the higher coefficient of variation at lower HBeAg levels. The intra-assay variation
was less than 5% (range 0.54 – 2.7%) for all samples (results not shown).
Each day that clinical samples were tested, aliquots of the diluted reference
sample (1 in 16 – 1 in 2048) were used to generate a standard curve. Linear regression
was used to convert the assay result into PE IU/mL for each sample. Where samples fell
outside the linear range of the assay (approximately 0.5 – 90 PE IU/mL, but defined by
the standard curve for each assay run), serial dilution (1 in 10, 1 in 100, 1 in 1000, ± 1 in
10,000) was performed using the ARCHITECT HBsAg Manual Diluent. Non-reactive
human serum was used as the negative control.