This Phase II proposal follows successful Phase I studies and describes the development, optimization, scale up, and utility testing of an efficient and cost effective technology to produce and purify high quality and affordable customized scFv antibodies for use in multiplex assays such as antibody arrays. The limited quality and number of reagents available for the construction of antibody micro arrays makes them costly to produce and unreliable to use. Polyclonal and hybridoma derived monoclonal antibodies, frequently don't have the characteristics suitable for use in these micro arrays nor are amenable to molecular evolution for higher affinity and the ability to capture native proteins from solution whilst conjugated to the surface of the micro array. This proposal utilizes yeast display technology to identify, develop, and optimize production of antibody molecules that function under the conditions of the micro array assay, incorporate these into micro arrays and further validate these by using solid tumor samples. The first targets we have selected are a series of receptor tyrosine kinases and their cognate ligands that are known to be involved in multiple types of cancer and for which therapeutics are currently either marketed or in development. The development of high quality antibody reagents for use in the construction of micro arrays will have broad implications in making diagnostic and research tools. ? ?
