A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines. Application to wart sections.
We compared nine different techniques for the detection of biotinylated DNA-DNA HPV hybrids on HeLa cells with 10-50 copies of HPV 18 DNA per cell. CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions (Tm = -17 degrees C) containing biotinylated DNA probes (cloned HPV types 1, 2, 6, 11, 16 and 18), at 90 degrees C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50 degrees C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37 degrees C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody - a biotinylated goat anti-rabbit antibody - a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin - anti-avidin goat antibody or anti-streptavidin rabbit antibody - a biotinylated anti-goat (or anti-rabbit) antibody - a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.
Guérin-Reverchon
I
I
INSERM U209, CNRS UA 601, Clinique Dermatologique, Hôpital E. Herriot, Lyon, France.
Chardonnet
Y
Y
Chignol
M C
MC
Thivolet
J
J
D002277:Carcinoma / Q000382:microbiology*; D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis*; D005615:Freezing; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D010232:Paraffin; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral; D010232:Paraffin