A simplified and rapid staining method for the HeLa x skin fibroblast human hybrid cell neoplastic transformation assay

Mendonca, M.S.; Antoniono, R.J.; Sun, C.; Redpath, J.L.

Radiation Research 131(3): 345-350


ISSN/ISBN: 0033-7587
PMID: 1279739
DOI: 10.2307/3578426
Accession: 006966977

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A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa X skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precepitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce that IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs .gamma. radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.