A NOVEL QUANTITATIVE IMMUNOASSAY SYSTEM FOR P53 USING ANTIBODIES SELECTED FOR OPTIMUM DESIGNATION OF P53 STATUS

Aim--To develop a highly sensitive and specific enzyme linked immunoso rbent assay (ELISA) system for analysis of p53 protein in cancer lysat es. Methods--The anti-p53 monoclonal antibodies DO7, 1801, BP53.12, an d 421, and anti-p53 polyclonal antiserum CM1 were assessed by immunohi stochemistry and western blot analysis to identify those most suitable for determining p53 status of cancer cells. Antibodies with desired c haracteristics were used to develop a non-competitive sandwich type EL ISA system for analysis of p53 expression in cancer cytosols. Using th e ELISA, p53 protein concentrations were measured in a small series of breast cancers, and the quantitative values compared with p53 immunoh istochemical data of the same cancers. Results--DO7 and 1801 gave the most specific and reliable results on immunohistochemistry and western blot analysis. Using these two antibodies, a noncompetitive sandwich type ELISA system was developed to analyse p53 quantitatively. Analysi s of the breast cancer series showed a good correlation between immuno histochemistry and the ELISA-tumours were generally positive using bot h techniques. Discrepancies were noted however: some cancers were immu nohistochemically negative but ELISA positive. One explanation for thi s may be that the ELISA is more sensitive than immunohistochemistry. C onclusion--The p53 ELISA system is a non-competitive double monoclonal antibody sandwich method, using DO7 and 1801 which have been shown to be highly specific for p53 protein by immunohistochemistry and wester n blot analysis. The lower threshold of the assay is 0.1 ng/ml analyte in an enriched recombinant p53 preparation. As p53 is now regarded as a protein associated with prognosis in breast and other cancers, the assay may have clinical applications.