Folie 1

1
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
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 1100 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
37C, real-time PCR of X-region
115
89
81
0.01
1
lt0.1
5
Sucrose gradient centrifugation of reference
samples for HBV particles
SW 41, 25,000 rpm, 16h, 10C, 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 1.5 ml
30 1 ml
40 2 ml
50 1 ml
60 1 ml
6010 KBr 1 ml
6
Banding of HBV DNA together HBV particles
25,000 rpm, 16h, 10C
7
Conclusion

• All analysed reference samples contained HBV DNA
  in virus- associated form