We propose to work collaboratively with investigators funded through this R21 mechanism to develop a new protein detection technology, the nano-capillary immunoassay. This technology will result in significant advances in protein detection and analysis with utility for both basic research and as a clinical diagnostic device. Our group, consisting of researchers at Lawrence Berkeley National Laboratory and Cell Biosciences, Inc., has the multidisciplinary expertise that this project requires. We will use CD44 in breast cancer as our model system to develop and optimize the nano-capillary immunoassay to reach optimal sensitivity (fg/?L), accuracy, and reproducibility. CD44 is an ideal model protein that will, if we are successful, demonstrate the potential of the technology to identify multiple splice variants and post-translational modifications (e.g., glycosylation, phosphorylation, and proteolysis) in cancer cell lines, tumor samples, and blood serum. Through this funding we will engineer improvements in protein capture chemistry, analyte detection, background reduction, and temperature control to increase sensitivity. We will also develop a new detection system to enable multiplexed analysis of several analytes in a single assay. Standardized protocols will be developed for optimal protein extraction from a range of biologic samples coupled with full automation resulting in high throughput and reproducibility. These approaches will yield a systematic and quantitative approach to protein analysis. This technology fulfills multiple goals of the RFA. 1) The nano-capillary assay offers unbiased detection of protein isoforms and post-translational modifications using a single detection reagent that will produce more complete protein profiles than can be achieved with standard protocols. 2) It will advance our knowledge of the molecular nature of cancer and provide a platform for the discovery of new biomarkers. 3) It has the potential for identifying undiscovered forms of existing biomarkers that may enhance their application as clinical diagnostics. The ultimate goal is to develop a technology to propel cancer biomarker discovery and enable early detection of breast cancer. ? ? ? ? ?
