Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

Chie Miyazaki, Yoshitaka Iba, Yukio Yamada, Haruo Takahashi, Jun Ichi Sawada, Yoshikazu Kurosawa

Research output: Contribution to journalArticlepeer-review

35 Citations (Scopus)


The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the V(H) domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire V(H-) coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.

Original languageEnglish
Pages (from-to)407-415
Number of pages9
JournalProtein Engineering
Issue number5
Publication statusPublished - 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology


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