Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase methods were used, the RAP (RA, ras; P, peptide) MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as well as normal mammary and colonic epithelia examined. We have used the RAP MAbs to define ras p21 protein expression in a spectrum of clonic disease states. Immunohistochemical analyses of individual cells within tissue sections reveal differences in ras p21 expression in colon carcinomas compared with normal colonic epithelium, benign colon tumors and inflammatory or dysplastic colon lesions. Our data suggest that ras p21 expression is correlated with depth of carcinoma within the bowel wall, and is probably a relatively late event in colon carcinogenesis. We have recently developed a quantitative lequid competition RIA for ras p21 using MAb Y13259. By the use of a standard of pure recombinant ras p21, we are now able to detect p21 at fM levels, and to determine the number of molecules of p21 per cell. The concomitant use of this quantitative RIA and immunohistochemical analyses of normal, dysplastic and inflammatory disease states, as well as carcinomas, should lead to a better understanding of the role of the ras gene in the genesis of these lesions.
