A standardized Antibody Microarray-based System for the Quantitative Measurement

DESCRIPTION (provided by applicant): The genesis of cancer involves several genetic changes that culminate in uncontrolled cellular proliferation. Importantly, there is evidence that splice variants of certain genes, and their respective ratios are significantly changed in the cancerous state. However, one of the major obstacles in elucidating the significance of cancer-associated splice variants is the lack of a systematic technology that can reliably detect and quantitate them at the protein level. Epitome Biosystems has developed a new method for quantitative proteomics that utilizes antibodies developed against unique continuous linear peptide sequences (EpiTagTMs) found within every protein. By utilizing a sandwich assay format with the EpiTagTM technology, Epitome has developed a systematic methodology that allows for the detection of virtually any splice variant within the proteome. Once the proteome is denatured and digested, each individual isoform generates a unique signature that can be quantified based upon novel antibody sandwich pair formation. The specific aims of this application are to; i) demonstrate the application of the EpiTagTM technology for the detection of splice variants; ii) develop and qualify an antibody array for the measurement of protein isoforms from two genes, Bcl-x and CD44; and iii) demonstrate the quantification of isoform levels for Bcl-x and CD44 from cultures of human cell lines and human serum samples. These aims in Phase I will allow for the utilization of a new technology that will detect cancer-related splice variants from complex biological samples. In Phase II, we will comprehensively assess these isoform expression patterns across numerous human breast cancer cell lines and human cancer tissue samples. We would then like to expand our measurement capability to protein isoforms from 20 gene families, and investigate two additional cancers such as lung and ovarian. The expansion of the program to include splice variants from multiple genes and across two different cancers will undoubtedly demonstrate the broad-based application of Epitome's technology for splice variant detection. The continued development and expansion of this technology holds great promise for new insights into cancer biology. 7. Project Narrative The proposed project seeks to apply an emerging antibody array technology for the detection of cancer-related splice variants from complex biological samples. The continued development and expansion of this technology holds great promise for new insights into cancer biology that can impact human health through better detection of disease and ultimately enhanced treatment options