Phage-display selection of antibodies to the left end of CTX3C using synthetic fragments

Yoko Nagumo, Hiroki Oguri, Kouhei Tsumoto, Yumi Shindo, Masahiro Hirama, Takeshi Tsumuraya, Ikuo Fujii, Yoshihisa Tomioka, Michinao Mizugaki, Izumi Kumagai

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

Ciguatoxins (CTXs) are a family of toxins that contaminate a variety of fish and cause ciguatera seafood poisoning. The limited availability of CTXs from natural sources has hampered preparation of antibodies and, thus, the development of immunoassays for these toxins. In the current studies, we utilized synthetic fragments as haptens to prepare antibodies against CTX3C, a congener of CTXs, thereby avoiding the problem of its scarcity. Synthetic ABC-ring fragment (ABC) of CTX3C was conjugated with keyhole limpet hemocyanin (KLH) and immunized on mice. Phage-displayed antibodies were first screened based on affinity to a soluble biotin-linked ABC-ring fragment that was captured on streptavidin-linked magnetic beads. The beads were then eluted with the ABCD-ring fragment (ABCD), and recovered phages were amplified. This elution with a synthetic fragment allowed the preparation of antibodies to ABCD as well as to ABC. Three antibodies with affinities of Kd∼10-5 M for ABCD were selected and prepared as soluble recombinant Fabs (rFabs). One rFab, 1C49, bound to CTX3C itself, although the binding affinity for CTX3C was weaker than for ABCD.

Original languageEnglish
Pages (from-to)137-146
Number of pages10
JournalJournal of Immunological Methods
Volume289
Issue number1-2
DOIs
Publication statusPublished - 2004 Jun

Keywords

  • 1-Hydroxybenzotriazole
  • Antibody
  • BSA
  • CTX
  • Ciguatoxin
  • DMF
  • ELISA
  • HOBt
  • Hapten
  • KLH
  • N, N-dimethyl formamide
  • Phage display
  • Synthetic fragment
  • bovine serum albumin
  • ciguatoxin
  • enzyme-linked immunosorbent assay
  • keyhole limpet hemocyanin
  • rFab
  • recombinant Fab

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Fingerprint

Dive into the research topics of 'Phage-display selection of antibodies to the left end of CTX3C using synthetic fragments'. Together they form a unique fingerprint.

Cite this