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Voss, Edward W., Jr. Weidner, Karla Marie 2014-12-17T23:15:37Z 2014-12-17T23:15:37Z 10000-01-01 1992 1992 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. Made available in DSpace on 2014-12-17T23:15:37Z (GMT). No. of bitstreams: 1 9305728.pdf: 6063101 bytes, checksum: 1fba4fc61c86c15e3df6313d7289a0d6 (MD5) Previous issue date: 1992 Embargo set by: Seth Robbins for item 72692 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs U of I Only 150 p. Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992. http://hdl.handle.net/2142/72524 (UMI)AAI9305728 Chemistry, Biochemistry Health Sciences, Immunology Characterization and Analysis of Anti-Metatype Antibodies text Microbiology Microbiology University of Illinois at Urbana-Champaign Dissertation Ph.D.