Antibody affinity maturation in vitro using unconjugated peptide antigen

Hiroto Iwai, Bengü Öztürk, Masaki Ihara, Hiroshi Ueda*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

18 Citations (Scopus)


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 VH-displaying phages for superior binders to the immobilized VL fragment in the presence of limited amount of antigen peptide, VH 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 without using conjugated antigens, and especially useful in the affinity maturation of antibodies whose Fv has limited stability in the absence of antigen.

Original languageEnglish
Pages (from-to)185-193
Number of pages9
JournalProtein Engineering, Design and Selection
Issue number4
Publication statusPublished - Apr 2010
Externally publishedYes


  • affinity maturation
  • antibody library
  • hapten
  • open sandwich selection
  • phage display


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