Method for the validation by Western analysis of affinity reagents against post-translationally modified proteins.

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ABSTRACT Antibodies (Abs) are commonly employed in biomedical research to identify and quantify target proteins. However, the specificity, sensitivity and reliability of these Abs can be variable, which is a significant concern in the scientific community. The use of an incorrect Ab can result in misleading or inaccurate results, impeding progress in various areas of biomedical research. In Phase I of this project, we developed tools to validate the specificity of commercial Abs used to detect phosphorylated sites in proteins in a method we developed and published that we named MILKSHAKE. We will present some of these results from this Phase I effort, which demonstrates the critical need to address this concern. This proposed SBIR Phase II project aims to both extend and commercialize the Phase I effort. We will continue the Ab validation work initiated in Phase I by extending the analysis to other types of post-translational modifications (PTMs) of proteins in Western analysis and flow cytometry. This Phase II project also seeks to improve and scale this pipeline further, and to develop new methods for validating Abs against glycosylated proteins, which are often dynamic and challenging to detect using traditional techniques. We will also employ an innovative tool for measuring Ab promiscuity using a technology we term ‘Sundae’ to better investigate the specificity of the Ab paratope. Sundae will be demonstrated for two therapeutic Abs. The Phase II project has several key objectives, including the development of new Ab-specific assays and the establishment of a centralized database for sharing validated Ab data. The project team comprises experts in Ab validation, proteomics, and molecular biology, and will collaborate closely with our current and future industry partners and academic researchers to ensure our success.