Colorimetric assay system for screening antiviral compounds against hepatitis B virus.
A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 microliters of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.
Sudo
K
K
Rational Drug Design Laboratories, Fukushima, Japan.
Konno
K
K
Shigeta
S
S
Yokota
T
T
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D003124:Colorimetry / Q000379:methods*; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis / Q000187:drug effects*; D004353:Drug Evaluation, Preclinical; D006510:Hepatitis B Antibodies / Q000276:immunology; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D008969:Molecular Sequence Data; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D063065:Organophosphonates; D009943:Organophosphorus Compounds; C059499:9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine; C053001:adefovir; C050127:9-(S)-(3-hydroxy-2-(phosphonomethoxy)propyl)adenine; D000225:Adenine