Antibody affinity maturation in vitro using unconjugated peptide antigen

Protein Engineering, Design & Selection : PEDS
Hiroto IwaiHiroshi Ueda

Abstract

Selection of antibody library in vitro is almost always performed on a certain solid-phase with immobilized antigen. However, for the selection of small molecule binders, conjugation of the antigen to a carrier molecule is indispensable, which often leads to the selection of unwanted binders such as conjugate-binders or those with insufficient specificity. Here we describe a rapid and efficient way to improve the affinity of an anti-small molecule antibody without antigen derivatization. The method is based on the open-sandwich (OS) principle, which utilizes the antigen-dependent stabilization of antibody variable domain Fv. We used an anti-osteocalcin C-terminal peptide Fv that showed a good response but with moderate sensitivity in OS ELISA as a model. By selecting PCR-randomized V(H)-displaying phages for superior binders to the immobilized V(L) fragment in the presence of limited amount of antigen peptide, V(H) mutants that show superior detection sensitivity in OS ELISA were obtained, and were characterized to retain improved antigen-binding affinity. Furthermore, saturation mutagenesis of a mutant resulted in further improvement in sensitivity. This 'OS-selection' will be the first to select anti-small molecule antibodies...Continue Reading

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Citations

Jan 27, 2016·Journal of Bioscience and Bioengineering·Jinhua DongHiroshi Ueda
Jan 25, 2012·Journal of Immunological Methods·Jinhua DongHiroshi Ueda
Sep 13, 2013·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Sharif HasanHiroshi Ueda
Nov 25, 2020·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Yoshitaka HaradaHiroshi Ueda
Aug 11, 2019·Ecotoxicology and Environmental Safety·Yang CongJinhua Dong

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