Properties of immunoglobulin Fv domains: Influence of valence and protein dynamics on antibody/antigen interactions
Mallender, William David
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Permalink
https://hdl.handle.net/2142/20562
Description
Title
Properties of immunoglobulin Fv domains: Influence of valence and protein dynamics on antibody/antigen interactions
Author(s)
Mallender, William David
Issue Date
1996
Doctoral Committee Chair(s)
Voss, Edward W., Jr.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Health Sciences, Immunology
Language
eng
Abstract
Anti-metatype antibodies are immunoglobulins specific for liganded antibody active sites that, upon binding, delay dissociation of primary ligand resulting in augmented primary antibody affinity for ligand. Anti-metatype antibodies, both monoclonal and polyclonal, specific for the 4-4-20/fluorescein complex, have been generated and extensively studied. Preliminary studies identified reduced variable domain dynamics as the reason for 4-4-20 affinity for fluorescein enhancement due to anti-metatype antibody binding. Differences in activity levels between monoclonal and polyclonal anti-metatype reagents, however, implicated valence, specificity and affinity for generating a functional anti-metatype stabilization effect. In order to investigate this hypothesis, a bivalent-bispecific single-chain antibody (BiSCA), consisting of the SCA derivatives of IgG 4-4-20 and IgG 04-01 (anti-ssDNA), was constructed and characterized as a prelude to construct novel BiSCA anti-metatype proteins. Following primary structure determination and failure to successfully produce active anti-metatype SCA molecules from candidate monoclonal antibodies, a bispecific anti-metatype monoclonal antibody was produced by chemical cross-linking methods. Results from studies comparing the activity of various anti-metatype antibody reagents confirmed the roles of valence and multispecificity in the anti-metatype stabilization effect. In addition, further analyses of the effect of anti-metatype stabilization on antibody variable domains was accomplished by construction and characterization of the Fv derivative of 4-4-20. This active site derivative protein, unlike SCA, lacked the interdomain linker peptide, permitting examination of the effect of anti-metatype stabilization on variable domain association. Such studies yielded significant information concerning the dependence of antibody active site dynamics on high affinity antigen binding and the importance of antibody constant domains on the maintenance of these dynamics. Collectively, studies of this nature could be applied to other protein systems where multivalent protein subunit associations are employed for effective ligand binding.
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