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Title:Affinity Maturation of the D1.3 Antibody Using Yeast Surface Display and Flow Cytometry
Author(s):VanAntwerp, Jennifer Jewett
Doctoral Committee Chair(s):Wittrup, K. Dane
Department / Program:Chemical Engineering
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Abstract:Different methodologies were explored for improving YSD affinity maturation of anti-protein antibodies, using scFv D1.3. Using equilibrium sorted YSD with the D1.3/M3 antibodies, a single-pass enrichment factor of 125-fold (+/-65-fold) was achieved, indicating excellent differentiation between clones of only slightly different affinity. Optimal equilibrium affinity screening of a randomly mutated D1.3 library was performed with and without an osmotic stressor present in the binding reactions. Osmotic stress yielded a more diverse set of mutant clones from the screen, and recombination of selected mutations using site-directed mutagenesis produced a four-fold higher affinity mutant (MEC1). MEC1 was randomly mutagenized and screened by kinetic selection, identifying a large number of improved mutants. In both screens, library size was approximately 5 x 106, and mutagenesis was by error-prone PCR of the entire scFv gene. This affinity compares favorably to the highest affinities attained previously with phage display for anti-protein antibodies, without the necessity for large libraries or site-directed saturation mutagenesis.
Issue Date:1999
Description:165 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1999.
Other Identifier(s):(MiAaPQ)AAI9953167
Date Available in IDEALS:2015-09-25
Date Deposited:1999

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