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Analysis of Solute-Binding Agent Interactions by High-Performance Affinity Chromatography and Sandwich Immunoassay
Information on solute-binding agent interactions is essential to determine the correct dosage of a medicine, for screening micropollutants in the environment, and for detecting viruses. This dissertation covered studies that designed and optimized high-performance affinity chromatography (HPAC) techniques such as ultrafast affinity extraction (UAE) and zonal elution to investigate the binding interactions of drugs with serum proteins and humic acids, respectively, and to create a sandwich immunoassay for detecting antibodies against COVID-19. The first and second chapter provide an introduction to the different formats of HPAC and how they can be utilized to study the thermodynamics and kinetics of solute-binding agent interactions. The third and fourth chapters describe the use of UAE and affinity microcolumns for measuring free drug fractions, equilibrium constants, and rate constants of normal or modified human serum albumin (HSA) with warfarin. It was observed that the binding of warfarin to Sudlow site I of HSA was impaired due to the formation of advanced glycation end products by methylglyoxal during type-II diabetes but not by modification caused by glyoxal. The fifth chapter describes the development of humic acid microcolumns made by non-covalent entrapment and used to screen binding by this agent with various pharmaceuticals found in environmental water by using HPAC and zonal elution. Several parameters were optimized to develop these microcolumns and to study the thermodynamics of drug-humic acid interactions. The sixth chapter describes the design and validation of a magnetic bead-based sandwich immunoassay, using the recombinant S1 subunit of spike protein for the detection of antibodies against COVID-19 in a tube format. The proposed assay could be used for the detection of multiple types of antibodies against spike protein’s S1 subunit within 5 minutes. The seventh chapter describes the use of chromatographic theory to predict the general conditions that are needed in UAE to obtain free solute fractions that provide accurate and precise measurements of equilibrium constants and rate constants for solutes and binding agents.
Iftekhar, Sazia, "Analysis of Solute-Binding Agent Interactions by High-Performance Affinity Chromatography and Sandwich Immunoassay" (2022). ETD collection for University of Nebraska-Lincoln. AAI29998365.