Comparison of different in situ hybridization techniques for the detection of human papillomavirus DNA in cervical smears.

Different hybridization methods were used for detection of human papillomavirus (HPV) DNA in cervical smears. The results obtained by filter in situ hybridization (FISH) are consistent with most of the reports recently published. To overcome the unsatisfactory limitations of this method, especially the difficulties to distinguish clearly between positive and negative signals, we developed an in situ hybridization protocol using a cytospin and 35S-labelled as well as biotinylated DNA-probes. For direct comparison of different methods, the samples were obtained from two groups of patients. One group were women with reiterated Papanicolaou smears III, IV; the other were women with reiterated Pap III, IV and additional histological scoring. In all cases but one, the different methods used have shown the same results. In one case the hybridization on slides using 35S-labelled as well as biotinylated probes gave a negative result, whereas the FISH method using a 32P-labelled probe allowed to detect of HPV 16 and 18 DNA only when more than 1 x 10(6) cells were present per filter. Our data demonstrate that in situ hybridization on slides is a specific and sensitive technique, which enables a clear distinction between positive and negative results using a small number of cells and which, especially with biotinylated probes, is suitable for application in routine work.

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.