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Development of sandwich microcolumns for use in rapid chromatographic immunoassays

William Alex Clarke, University of Nebraska - Lincoln


The work in my dissertation involves the development of microcolumns for performing rapid chromatographic immunoassays. The areas of study involved the development of microcolumns with lengths of less than 1 mm, optimization of antibody immobilization to chromatographic supports, and the separation of free warfarin from serum protein bound warfarin. In addition, sandwich microcolumns were used in the development of a rapid chromatographic immunoassay for free thyroxine (T4) in serum. ^ Part one deals with optimizing the amount of antibody that can be immobilized to a chromatographic support. This study examined several factors, including immobilization of Fab fragments versus entire antibodies, the use of general or site-specific attachment, and differences between the immobilization of antibodies to silica or other commercially available supports. ^ Part two explores the development of microcolumns with sub-millimeter lengths and the reproducibility of making these columns. Reproducibility of the method was determined by packing hemoglobin microcolumns and analyzing each microcolumn for total protein content. Studies were performed to determine the feasibility of using these columns for rapid chromatographic immunoextractions under both diffusion-limited and adsorption-limited conditions. ^ In part three, the use of microcolumns to separate the free and protein-bound fractions of warfarin is explored. The extraction of warfarin in a sub-second time frame was optimized in order to minimize the interference from protein bound warfarin released during this extraction. Microcolumn immunoextraction coupled with restricted-access media chromatography was used to separate the free and bound warfarin and determine the amount of free warfarin in the sample. The experimental results were then compared with mathematically predicted values as well as the results of a computer simulation. ^ The last part of this dissertation involves the development of a chromatographic immunoassay for free T4 in serum. The analysis was performed using a displacement immunoassay format with chemiluminescent detection and a labeled analog. The reproducibility of the method was established and a standard curve was obtained using various T4 concentrations. Finally, the amount of free T4 in a serum sample was determined. ^

Subject Area

Chemistry, Analytical

Recommended Citation

Clarke, William Alex, "Development of sandwich microcolumns for use in rapid chromatographic immunoassays" (2000). ETD collection for University of Nebraska - Lincoln. AAI3070125.