Use of digoxigenin-labelled probes for the quantitation of HBV-DNA in antiviral drug evaluation

The use of digoxigenin-labelled probes was studied for quantitation of HBV- DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes wer e generated either via incorporation of dig-dUTP in a polymerase chain reac tion (PCR) or a random priming reaction. Using the PCR-labelled probe (deli neating a 523 bp fragment in the core gene of the HBV) as little as I pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hyb ridized membrane. A close correlation (r = 0.95) was found between the amou nt of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybr idized to its target DNA. By using a probe that was labelled with digoxigen in via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a P-32-l abelled probe, generated in parallel by random priming, allowed the detecti on of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generate d digoxigenin-labelled probe proved to be useful for antiviral drug evaluat ion, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatom a cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penci clovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were co mparable to those reported in the literature. The use of digoxigenin-labell ed probes thus appears to be a simple, convenient, rapid, reliable and non- radioactive method for use for anti-HBV screening. In addition, and in cont rast to P-32-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes partic ularly attractive for large-scale antiviral drug evaluation purposes. (C) 1 999 Elsevier Science B.V. All rights reserved.