In situ hybridization detection of single-copy human papillomavirus on isolated cells, using a catalyzed signal amplification system: GenPoint.
The performance and drawbacks of GenPoint, 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 hybridization with biotinylated DNA probes. For this aim, formalin-fixed cell deposits from carcinoma cells of the uterine cervix, CaSki, SiHa, and HeLa, containing, respectively, 600 copies of HPV DNA type 16, 1-2 copies of HPV DNA type 16, and 10-50 copies of HPV DNA type 18, were used, and the GenPoint method (consisting of successive incubations with peroxidase-conjugated streptavidin, biotinyl tyramide, and peroxidase-conjugated streptavidin) was compared to immunoenzymatic revelation procedures involving either a one-step reaction (streptavidin-alkaline phosphatase or streptavidin-peroxidase), or a three-step reaction (anti-biotin mouse monoclonal antibody, rabbit anti-mouse antiserum, and mouse APAAP complex). In these conditions, after analysis with a bright-field microscope, GenPoint appeared the most sensitive method of revelation, easily allowing detection of 1-2 copies of HPV DNA on isolated cells by in situ hybridization.
Lizard
G
G
Laboratoire de Biochimie Médicale, CHU/Hôpital du Bocage, Inserm U498, BP 1542, 21034 Dijon Cedex, France. Gerard.Lizard@u-bourgogne.fr
Démares-Poulet
M J
MJ
Roignot
P
P
Gambert
P
P
D019921:Biotinylation; D002277:Carcinoma / Q000821:virology; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis*; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015344:DNA Probes, HPV; D004279:DNA, Viral