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

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

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

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%

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

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

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