Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.
A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.
D004259:DNA-Directed DNA Polymerase / Q000097:blood*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D017245:Foscarnet; D006509:Hepatitis B / Q000201:enzymology; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D019384:Nucleic Acid Synthesis Inhibitors; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*
D003845:Deoxycytosine Nucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D009943:Organophosphorus Compounds; C024107:2'-deoxycytidine 5'-triphosphate; D017245:Foscarnet; D004259:DNA-Directed DNA Polymerase; D010746:Phosphonoacetic Acid