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 immunoassayis 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-HBe-negative 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 1and 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. / COI of initial assay (1:40 dilution factor) / COI of repeated assay (undiluted) / COI of repeated assay (further 1:4 dilution factor) / Conversionfrom COI to PEIU/mL (COI*dilution factors*0.222) / HBeAg level(PEIU/mL)
1 / <1 / 5 / N/A / 5*1*0.222 / 1.11
2 / 10 / N/A / N/A / 10*40*0.222 / 8.88
3 / >1000 / N/A / 750 / 750*4*40*0.222 / 26,640
* based on Wursthorn K et al. Development of a protocol for the quantitative determination of HBeAg using the Elecsys® HBeAg 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