In situ hybridization detection of single-copy human papillomavirus on isolated cells, using a catalyzed signal amplification system: GenPoint (TM)

The performance and drawbacks of GenPoint(TM), which is a catalyzed signal amplification system for immunohistochemistry, have been evaluated for its ability to reveal human papillomavirus (HPV) DNA detected by in situ hybrid ization with biotinylated DNA probes. For this aim, formalin-fixed cell dep osits from carcinoma cells of the uterine cervix, CaSki, SiHa, and HeLa, co ntaining, respectively, 600 copies of HPV DNA type 16, 1-2 copies of HPV DN A type 16, and 10-50 copies of HPV DNA type 18, were used, and the GenPoint (TM) method (consisting of successive incubations with peroxidase-conjugate d streptavidin, biotinyl tyramide, and peroxidase-conjugated streptavidin) was compared to immunoenzymatic revelation procedures involving either a on e-step reaction (streptavidin-alkaline phosphatase or streptavidin-peroxida se), or a three-step reaction (anti-biotin mouse monoclonal antibody, rabbi t anti-mouse antiserum, and mouse APAAP complex). In these conditions, afte r analysis with a bright-field microscope. GenPoint(TM) appeared the most s ensitive method of revelation, easily allowing detection of 1-2 copies of H PV DNA on isolated cells by in situ hybridization. (C) 2001 Wiley-Liss, Inc .