TIME-RESOLVED IMMUNOFLUOROMETRIC ASSAY OF P53 PROTEIN

A common feature of human tumor tissue is mutant p53 protein accumulat ion. Here we evaluate a new ''sandwich'' immunoassay for p53 protein i ncorporating modifications to a previously reported method, including the use of microtiter plates coated directly with the anti-p53 monoclo nal antibody DO-1, a detergent- and mouse serum-containing sample dilu ent, and a labeled secondary antibody diluent containing goat serum. T he use of CM-1 antiserum to probe the immunocaptured p53 and the detec tion of bound complexes by a labeled secondary antibody allows couplin g to a time-resolved fluorescence detection system. The new assay yiel ded p53 concentrations comparable with those by the previous assay for breast tumor cytosols (n = 198), nondiseased breast tissues (n = 70), and five transformed cell lines, but showed differences in p53 values measured in sera from patients without cancer (n = 78). These serum d ifferences were found to reflect nonspecific interferences affecting t he original method, which implies that the new immunoassay has improve d specificity for serum p53 quantification.