Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto‐oncogene p21 products

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Abstract
The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. This gene family mediates transformation via (1) a point‐mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21. Group‐specific, type‐selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and protp‐oncogene products, have been developed. Using purified recombinant ras p21 from Escherichla coli expressing the full‐length T24 mutant human Harvey‐ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E. coli expressing the point‐mutated or proto‐ras p21 and (2) mammalian cell lines of human and murine origin. Two of the RIAs developed can be termed group‐specific in that they have the ability to detect the point‐mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type‐selective, since it detects an antigenic determinant located primarily on the Harvey ras p21. All 3 RIAs are interspecies‐specific since they are able to detect ras p21 in rodent as well as human cells. The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression. These assays, used in conjunction with specific cDNA probes to identify specific ras proto‐oncogenes or point‐mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and transformation.