Date of Award
Doctor of Philosophy
Rodney R. Walters
Many aspects of the application of High Performance Affinity Chromatography (HPAC) to bioanalysis are addressed. The initial study demonstrates the quantitative determination of IgG from serum using HPAC. Columns were prepared by immobilizing Protein A from Staphylococcus aureus on 10 m diol-bonded silica. Immunoglobulin (Ig)-containing samples were injected into the column at pH 7 and the non-retained components eluted. The retained proteins were then eluted by a step change to pH 3. The retention characteristics of the Protein A column were studied with the chromatography of IgG, IgA, and IgM as well as electrophoresis and Ouchterlony diffusion of retained and non-retained serum fractions. Retention of IgG1, IgG2 and some IgA was shown. IgM, some IgA and albumin were not retained. Analysis of reference serum without pretreatment was performed using IgG standards. Good agreement was observed between the IgG concentrations measured by this method and by radial immunodiffusion;In the second investigation, the immobilization and activity of protein ligands on silica supports were studied. The activation of diol-bonded silica with 1,1[superscript]'-carbonyldiimidazole and the coupling of protein ligands were examined as a function of activation conditions, coupling time, buffer, salt and pH. Coupling yields for small amines and for several proteins were found to be relatively insensitive to buffer and pH over the range from 4 to 8. The effect of immobilization on ligand activity was random;The dissociation of IgG from immobilized Protein A was examined in the final study. For this work the Peak Decay (PD) kinetic model was used. The derivation and a computer simulation of the model are reviewed. The application of the computer simulation for the estimation of the chromatographic parameters required to produce the PD effect is demonstrated. Using the PD model, the native dissociation rate was found to be 0.008 sec[superscript]-1. Low pH denaturing elution was also studied with the model. A dissociation rate of 1.2 sec[superscript]-1 was observed. The activation parameters of both the naturing and denaturing dissociation processes were determined. The suitability of the PD model for the study of protein-protein interactions as well as high affinity biocomplexes (K[subscript] eq < 10[superscript]6) is indicated.
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Samuel C. Crowley
Crowley, Samuel C., "Preparation and characterization of Staphylococcal protein A adsorbents for high performance affinity chromatography " (1988). Retrospective Theses and Dissertations. 9333.