Work performed during the Phase I of this project has demonstrated that formamide induces selective denaturation of DNA in apoptotic cells. This novel effect of formamide on the stability of apoptotic DNA combined with the detection of denatured DNA with monoclonal antibody (MAb)against single-stranded DNA, has made it possible to develop a sensitive and specific assay for the identification of apoptosis. The major goal of the proposed Phase II project is to develop our formamide-MAb assay for possible commercial applications. For this purpose, we will develop assay protocols for staining of histological sections from formalin-fixed paraffin-embedded archival material, frozen tissue sections and cytological preparations. Production of a formamide-MAb assay kit for commercial use will be the major goal of the Phase II project. Work proposed for the development of the kit will involve production and conjugation of anti-ssDNA MAb, selection of optimal protocols and reagents for the kit and evaluation of the stability of the kit components under variable storage and shipping conditions. To develop applications of formamide-MAb assay for the evaluation and prediction of therapeutic response in solid tumors, we propose to: a) evaluate relation between chemosensitivity and the induction of apoptosis, b) measure apoptosis in endothelium of tumors treated with antiangiogenesis factors and c) study apoptosis in tumors treated with specific apoptosis-inducing therapy (e.g. death ligand TRAIL). High-throughput ELISA based on formamide-MAb technique will be developed as a new and rapid method for high-volume screening of drugs for their apoptotic and anti-apoptotic activity.
Significant commercial applications could be expected for the proposed assay, which provides sensitive, specific and universal detection of apoptosis in cells and tissue sections and makes possible high-throughput screening of drugs for apoptotic and anti-apoptotic activity.
