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|Title:||Kinetic Analysis of High Affinity Polyclonal Anti-Fluorescyl Immunoglobulin-G Antibodies|
|Author(s):||Herron, James Nelson|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Mathematical and experimental tools for the measurement and assessment of association and dissociation kinetics in anti-fluorescyl antibodies were developed in these studies. An analytic procedure for the characterization of heterogeneous first order kinetics was derived. This procedure was based on Laplace transformation of the gamma distribution function, which yielded an exact rate law. Kinetic assays were developed for the measurement of both association and dissociation rates. Association rates were measured by following the increase in fluorescence quenching observed when anti-fluorescyl antibodies were added, in excess, to aqueous fluorescein. The reaction was made pseudo-first order to enable use of the same data processing routines for analysis of both association and dissociation kinetics. Dissociation rates were measured by following the increase in fluorescence intensity after the addition of excess 5-aminofluorescein to a population of fluorescyl-liganded antibodies. Since 5-aminofluorescein was a non-fluorescent competitive inhibitor of fluorescein, the increase in fluorescence intensity was directly attributable to the dissociation of fluorescyl-ligand.
Analysis of association reactions in both rabbit polyclonal and murine monoclonal populations of anti-fluorescyl-IgG antibodies revealed the presence of two-step association kinetics. This observation suggested that anti-fluorescyl-IgG antibodies intrinsically exhibited a two-step association mechanism. Dissociation kinetics were analyzed in both monoclonal and polyclonal anti-fluorescyl-IgG antibodies. As expected, monoclonal antibodies exhibited homogeneous dissociation kinetics. Polyclonal anti-fluorescyl antibodies, obtained from individual hyperimmune New Zealand White (NZW) rabbits, exhibited 3-4 different dissociative components. The component with fastest dissociation rate, was typically heterogeneous with an average dissociative lifetime of 8 seconds, where dissociative lifetime was defined as the reciprocal dissociation rate constant. Longer lifetime components exhibited lifetimes of several hundred seconds, and became progressively more homogeneous with increasing lifetime. Polyclonal anti-fluorescyl antibody preparations, obtained from 14 different hyperimmune NZW rabbits, were used to characterize the expression of anti-fluorescyl antibodies within the rabbit population. Analysis of dissociation kinetics revealed the presence of 4 distinct groupings of dissociative lifetimes, which were approximately Gaussian in their distribution. Average lifetimes of 8, 52.6, 313 and 1417 seconds were observed for the four groupings. Assuming identical association rates for the four groupings, their average affinities were separated by 1 Kcal/mole increments.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
|Date Available in IDEALS:||2014-12-14|