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Development of novel monoliths and affinity microcolumns for use in high performance affinity chromatography and pharmaceutical applications
High performance affinity chromatography (HPAC) is a useful tool for probing biological interactions such as the binding of an antibody with an antigen or the binding of a drug with a serum protein. This dissertation describes the development of affinity microcolumns for the rapid analysis of various analytes and the extension of some of these microcolumns for use with immunoaffinity chromatography (IAC). The first project that is presented concerns the use of an affinity microcolumn containing immobilized human serum albumin (HSA) for the development of a one-site immunometric assay. This approach employed a detection mode that made use of specific dyes that were conjugated to polyclonal anti-HSA antibodies. This method was able to provide a limit of detection for HSA of 0.03 ng/mL and a dynamic range that extended to 0.200 ng/mL HSA. This approach was employed with control serum samples and it was demonstrated that this approach could be used for the trace analysis of protein biomarkers in such a matrix. The next section of the dissertation focuses on the development of affinity microcolumns containing immobilized protein G for use as a secondary binding agent for antibodies and a platform for chromatographic immunoassays. The assay formats that were examined included two simultaneous injection methods (either column-based or solution-based) and a sequential injection format. Based on the analytical behavior of these methods, information was obtained that could be used to extend the use of protein G microcolumns and these assay formats to other proteins or biomarkers of clinical or pharmaceutical interest. The final section of this dissertation demonstrates the development of new, more rapid methods for the preparation of monolithic supports for use in affinity chromatography. These monoliths were based on co-polymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA). This study used affinity microcolumns that contained GMA/EDMA monoliths that were prepared using various initiation and polymerization conditions. These monoliths were evaluated for their ability to immobilize HSA and their ability to be used in binding studies involving R/S-warfarin.
Pfaunmiller, Erika L, "Development of novel monoliths and affinity microcolumns for use in high performance affinity chromatography and pharmaceutical applications" (2014). ETD collection for University of Nebraska - Lincoln. AAI3642826.