Selective Enrichment of Monospecific Polyclonal Antibodies for Antibody-Based Proteomics Efforts

J Chromatogr A. 2004 Jul 16;1043(1):33-40. doi: 10.1016/j.chroma.2004.06.008.

Abstract

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry*
  • Antibodies / isolation & purification
  • Antibody Specificity
  • Blotting, Western
  • Proteomics*

Substances

  • Antibodies