Supplementary 1
Quantitative HBeAg assay
HBeAg-positive samples were subjected to quantitative measurement using protocol recently developed by
Wursthorn et al (18) for the quantitative determination of HBeAg using the Elecsys® HBeAg
immunoassay (Roche Diagnostic GmbH, Manheim, Germany). The Elecsys® HBeAg immunoassay is an
electrochemiluminescence immunoassay (ECLIA) for qualitative detection of HBeAg in human serum and
plasma. A restricted range of Cut-off index (COI) has been validated to show linear association with
HBeAg titer, allowing this system to be used for quantification of serum HBeAg. This linearity was
established by measuring a range of serial dilutions of recombinant HBeAg using five different diluents
(HBeAg-negative/anti-HBe-negative diluent, Elecsys Universal Diluent, HBeAg-negative/anti-HBenegative human serum, horse serum and phosphate-buffered saline). Using the Paul Ehrlich Institute (PEI)
international reference standard serum, conversion factor was established as 4.5 COI for 1 PEIU/mL,
corresponding to 0.222 PEIU/mL for a COI of 1. An assay with an initial dilution step of 1:40 is considered
to provide accurate assessment of the majority of samples encountered in clinical practice. An algorithm
has been developed for the quantification of HBeAg using the Elecsys® HBeAg assay.
In this study, serum samples were diluted 1:40 in Elecsys Universal Diluent and assayed using a Roche
Cobas® e411 Immunoanalyzer following the manufacturer’s instructions. Resulting COIs between 1 and
1000 were converted to PEIU/mL by multiplying the COI values with the conversion factor 0.222 and the
dilution factor 40. Samples with COIs < 1 were re-assayed undiluted, and the resulting COIs were
multiplied with 0.222. Meanwhile, samples with COIs >1000 were further diluted and re-assayed, and the
resulting COIs were multiplied with the dilution factors (40 and the second dilution factor) and 0.222. An
example of calculating the results of HBeAg quantification is shown below:
Example of calculating results of HBeAg quantification using the Elecsys® HBeAg assay*
No.
1
2
3
COI of initial
assay (1:40
dilution factor)
<1
10
>1000
COI of repeated
assay (undiluted)
COI of repeated
assay (further 1:4
dilution factor)
5
N/A
N/A
N/A
N/A
750
Conversion from
COI to PEIU/mL
(COI*dilution
factors*0.222)
5*1*0.222
10*40*0.222
750*4*40*0.222
HBeAg level
(PEIU/mL)
1.11
8.88
26,640
* based on Wursthorn K et al. Development of a protocol for the quantitative determination of HBeAg using the Elecsys®
immunoassay. J Viral Hepat 2011;18(7):179-183
COI: Cut-off Index
PEI: Paul Erlich Institute, used as international reference standard for HBeAg level
HBeAg