Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

Exp Mol Med. 2013 Sep 27;45(9):e43. doi: 10.1038/emm.2013.83.

Abstract

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bispecific / immunology*
  • HEK293 Cells
  • Haptens / immunology*
  • Humans
  • Immunoblotting / methods*
  • Immunoprecipitation / methods
  • Rabbits
  • Single-Chain Antibodies / immunology

Substances

  • Antibodies, Bispecific
  • Haptens
  • Single-Chain Antibodies