Affinity Maturation of the D1.3 Antibody Using Yeast Surface Display and Flow Cytometry
VanAntwerp, Jennifer Jewett
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https://hdl.handle.net/2142/82476
Description
Title
Affinity Maturation of the D1.3 Antibody Using Yeast Surface Display and Flow Cytometry
Author(s)
VanAntwerp, Jennifer Jewett
Issue Date
1999
Doctoral Committee Chair(s)
Wittrup, K. Dane
Department of Study
Chemical Engineering
Discipline
Chemical Engineering
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
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.
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