SBIR-STTR Award

The development of sensitive, specific and universal assay is needed to determine the role of apoptosis in biology and in tumor cell response to chemotherapy. A novel anti-ssDNA MAb assay developed by the applicant is designed to meet these requirements. The assay is based on the staining of apoptotic cells with a new monoclonal antibody (MAb) highly specific for single-stranded DNA. Importantly, MAb assay detects apoptosis in tissue sections, for which the only other available histochemical marker (TUNEL) has low sensitivity and limited specificity. The major goal of this proposal is validation of a novel anti-ssDNA MAb apoptosis assay against other available tests: annexin V staining, activation of caspases, decrease in mitochondria membrane potential and DNA fragmentation (TUNEL staining and DNA ladder). Modified protocols, which make possible application of the technique to archival and biopsy material, will be developed. The assay will be tested in different models in order to prove universal application and to determine correlation with drug sensitivity: apoptosis induced by cytotoxic agents, hormones, ligation of FAS receptor, apoptosis in hematopoetic and epithelial cell lines and apoptosis in drug-sensitive and drug resistant cell lines and solid tumors. PROPOSED COMMERCIAL APPLICATION: To market apoptosis assay.

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. PROPOSED COMMERCIAL APPLICATION: 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