1. Characterisation of HBV DNA in reference preparations
SoGAT XXI, Brussels May 29, 2009
Characterisation of HBV DNA in reference preparations
Corinna Bremer, Dieter Glebe, Wolfram H. Gerlich
Institute for Medical Virology (IMV), Justus Liebig University Giessen, Germany
National Consulting laboratory for Hepatitis B und D
Nico Lelie
Chiron Novartis Vaccines and diagnostics, Suresnes France

2. Characterisation of HBV DNA in reference preparations
SoGAT XXI, Brussels May 29, 2009
Characterisation of HBV DNA in reference preparations
Methods described in:
Corinna Bremer et al., Transfusion, 2009

3. Origin of Reference Materials for HBV DNA
WHO: dilution of Eurohep standard reference 1,
genotype A2, HBs subtype adw,
lyophilised 5 x 105 IU
disolved in 0.5 ml H2O  5 x106 ge/ml
VQC : 3 x 108/9 ge/ml (1997 Sanquin)
lower in our PCR  2 x 108 ge/ml
genotype A2
VQC pasteurized: 1.2 x 107 ge/ml
1:100 dilution of VQC
ISS genotype D: 7,500 IU/ml  3.75 x 104 ge/ml
ISS = Istitute Superiore di Sanità
Conversion factor IU to ge = 5

4. Digestion of free cloned or virus encapsidated HBV DNA
200 µl serum, 500 Units DNAse (Benzonase), 1 h 37°C, real-time PCR of X-region
89%
115%
81%
0.01% 1%
<0.1%

5. Sucrose gradient centrifugation of reference samples for HBV particles
SW 41, 25,000 rpm, 16h, 10°C, fractions of 0.5 ml,
Inhouse real-time PCR of the X-region
% sucrose
volume
Reference sample
0.2 ml 5
1 ml 10
1.5 ml 20 30 40
2 ml 50 60
60+10% KBr

6. Banding of HBV DNA together HBV particles
25,000 rpm, 16h, 10°C

7. Conclusion
All analysed reference samples contained HBV DNA in virus- associated form