A single-step “breeding” generated a diagnostic anti-cortisol antibody fragment with over 30-fold enhanced affinity

Hiroyuki Oyama, Izumi Morita, Yuki Kiguchi, Tomomi Morishita, Sakiko Fukushima, Yuki Nishimori, Toshifumi Niwa, Norihiro Kobayashi

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Cortisol levels in bodily fluids represent a useful index for pituitary−adrenal function, and thus practical anti-cortisol antibodies are required. We have studied “antibody-breeding” approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A “wild-type” single-chain Fv fragment (wt-scFv; Ka, 3.4×108 M−1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for this antibody. Then, random point mutations were generated separately in VH or VL by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity (Ka 1.2×1010 M−1). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its VL sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose–response curves with measuring range ca. 0.03–0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.

Original languageEnglish
Pages (from-to)2191-2198
Number of pages8
JournalBiological and Pharmaceutical Bulletin
Volume40
Issue number12
DOIs
Publication statusPublished - 2017

Keywords

  • Affinity maturation
  • Antibody
  • Cortisol
  • Enzyme-linked immunosorbent assay
  • In vitro evolution
  • Single-chain Fv fragment

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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