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Journal of Protein Chemistry

, Volume 17, Issue 3, pp 245–254 | Cite as

Single-Chain Fv of Anti-idiotype 11-1G10 Antibody Interacts with Antibody NC41 Single-Chain Fv with a Higher Affinity than the Affinity for the Interaction of the Parent Fab Fragments

  • Peter Iliades
  • David A. Dougan
  • Geoffrey W. Oddie
  • Dennis W. Metzger
  • Peter J. Hudson
  • Alexander A. Kortt
Article

Abstract

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 μg/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer–dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr ≍ 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-1G10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.

Antibody anti-idiotype single-chain Fvs solution properties complexes with antigens binding affinities 

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Copyright information

© Plenum Publishing Corporation 1998

Authors and Affiliations

  • Peter Iliades
    • 1
  • David A. Dougan
    • 1
  • Geoffrey W. Oddie
    • 1
  • Dennis W. Metzger
    • 2
  • Peter J. Hudson
    • 1
  • Alexander A. Kortt
    • 1
  1. 1.CSIRO, Division of Molecular ScienceParkvilleAustralia
  2. 2.Department of MicrobiologyMedical College of OhioToledo

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