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Entrapment of Ligands in High-Performance Affinity Columns for Chromatographic Studies of Solute-Ligand Interaction and Use of Sandwich Immunoassay for Antibody Detection
Affinity chromatography is a form of liquid chromatography that employs a biologically-related binding agent as the stationary phase. This technique exploits the selective and reversible binding that occurs in many biological interactions to isolate and purify specific targets. Affinity chromatography makes use of these systems by immobilizing one of a pair of interacting agents onto a chromatographic support. The agent immobilized onto the support provides a stationary phase with the ability to selectively retain the complementary target or solute. The integration of supports for high-performance liquid chromatography with affinity chromatography is known as high-performance affinity chromatography (HPAC). In recent decades, monolith materials have been utilized as supports in HPAC columns. The first chapter of this dissertation reviews the basic principles of affinity monolith chromatography and recent developments in this field. The elevated blood glucose level during diabetes can lead to modification of proteins like human serum albumin (HSA). This process is known as glycation. The second chapter examines how the modification of HSA to form advanced glycation end-products affects the binding of this protein to several first- and second-generation sulfonylurea drugs. This was done by immobilizing the modified HSA samples through non-covalent entrapment method in HPAC microcolumns. The third chapter compares several approaches to optimize the on-column entrapment procedure for proteins, using HSA as a model protein. The final conditions were then employed to entrap α1-acid glycoprotein (AGP) and to make mixed-mode supports containing HSA and AGP. The strength of the reversible interactions between drugs and humic acids in water is an important factor in determining the bioavailability of drugs in environmental water. The fourth chapter discusses the preparation of humic acid microcolumns prepared by entrapment and the use of these microcolumns to screen binding by drugs to humic acid. The fifth chapter describes the development of an assay for antibodies produced by the COVID-19 virus based on a fluorescence sandwich assay format and using small silica-coated magnetic beads.
Poddar, Saumen, "Entrapment of Ligands in High-Performance Affinity Columns for Chromatographic Studies of Solute-Ligand Interaction and Use of Sandwich Immunoassay for Antibody Detection" (2022). ETD collection for University of Nebraska - Lincoln. AAI29323342.