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Title:Characterization and Analysis of Anti-Metatype Antibodies
Author(s):Weidner, Karla Marie
Doctoral Committee Chair(s):Voss, Edward W., Jr.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
Health Sciences, Immunology
Abstract:Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity-labeled with fluorescein 5-isothiocyanate into rabbit elicited antibodies specific for the liganded conformation of 4-4-20. Termed anti-metatype antibodies, this polyclonal reagent also demonstrated the unique ability to delay the rate of dissociation of fluorescyl ligand from the active site of 4-4-20. This delay led to an artificial enhancement of the affinity of 4-4-20 for fluorescyl ligand. Utilization of a single chain derivative of 4-4-20 confirmed that anti-metatype reactivity was variable-region specific. Additionally, anti-metatype antibodies reacted with members of the 4-4-20 idiotype family, demonstrating varying degrees of affinity enhancement with idiotypically related molecules.
Anti-metatype antibodies significantly delayed dissociation of fluorescein structural analogues from the active site of 4-4-20. Fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carriers of increasing molecular weights to test whether anti-metatype antibodies could stabilize the liganded 4-4-20 molecule by binding at the mouth of the active site. Significant delays were also observed for these fluorescein-conjugated compounds in the presence of anti-metatype antibodies. These results suggest that the mechanism whereby anti-metatype antibodies stabilize the liganded 4-4-20 molecule cannot be attributed solely to steric hindrance of ligand release due to anti-metatype antibodies binding at the mouth of the active site. Solvent perturbation studies of 4-4-20 with deuterium oxide revealed a reduction in the dynamics of the entire 4-4-20 molecule in the presence of anti-metatype antibodies.
In an attempt to produce monoclonal anti-metatope antibodies, Armenian hamsters were immunized with both affinity-labeled and liganded 4-4-20. While no ligand-requiring anti-metatope antibodies were produced, seventeen monoclonal anti-4-4-20 antibodies were isolated. Hamster monoclonal antibodies were classified into 5 separate groups based on binding properties, including the ability of ligand to inhibit binding of anti-4-4-20 antibodies, binding to 4-4-20 idiotype family, binding to monoclonal antibody 04-01, and western blot analysis. Six of the seven non-ligand inhibitable monoclonal antibodies tested demonstrated the ability to enhance the affinity of 4-4-20 for its ligand. Additionally, two ligand inhibitable monoclonal antibodies demonstrated the unique ability to delay the rate of ligand association with 4-4-20. Results of studies with both polyclonal and monoclonal antibodies against the 4-4-20 variable domain demonstrate that this approach provides information about subtle structural differences between liganded and non-liganded antibodies, immunoglobulin molecules and their single-chain derivatives, and mutant and wild-type single-chain antibodies.
Issue Date:1992
Type:Text
Description:150 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992.
URI:http://hdl.handle.net/2142/72524
Other Identifier(s):(UMI)AAI9305728
Date Available in IDEALS:2014-12-17
Date Deposited:1992


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