Combined immunoaffinity cDNA-RNA hybridization assay for detection of hepatitis A virus in clinical specimens.
AUTOR(ES)
Jansen, R W
RESUMO
To apply cDNA-RNA hybridization methods to the detection of hepatitis A virus (HAV) in clinical materials, we developed a two-step method in which a microtiter-based, solid-phase immunoadsorption procedure incorporating a monoclonal anti-HAV capture antibody was followed by direct blotting of virus eluates to nitrocellulose and hybridization with 32P-labeled recombinant HAV cDNA. This immunoaffinity hybridization method is simple and involves few sample manipulations, yet it retains high sensitivity (10- to 30-fold more than radioimmunoassay) and is capable of detecting approximately 1 X 10(5) to 2 X 10(5) genome copies of virus. The inclusion of the immunoaffinity step removes most contaminating proteins and thus facilitates subsequent immobilization of the virus for hybridization. It also permits positive hybridization signals to be related to specific antigens and adds a level of specificity to the hybridization procedure. When the method was applied to 23 fecal specimens collected from individuals during week 1 of symptoms due to hepatitis A, 13 specimens were found to be reproducibly positive for HAV RNA by immunoaffinity hybridization, whereas only 11 contained viral antigen detectable by radioimmunoassay.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=271864Documentos Relacionados
- Dot blot hybridization assay of B19 virus DNA in clinical specimens.
- Specificity of dot hybridization assay in the presence of rRNA for detection of rotaviruses in clinical specimens.
- Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens.
- Evaluation of a radioactive rRNA:cDNA-hybridisation assay for the direct detection of Chlamydia trachomatis in urogenital specimens.
- Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay.