Towards a Human Proteome Atlas: High-Throughput Generation of Mono-Specific Antibodies for Tissue Profiling

Proteomics. 2005 Nov;5(17):4327-37. doi: 10.1002/pmic.200500072.

Abstract

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / chemistry*
  • Antibodies / isolation & purification
  • Antibody Specificity
  • Chromatography, Affinity
  • Humans
  • Immunohistochemistry / methods
  • Protein Array Analysis*
  • Proteome / chemistry*
  • Proteome / immunology
  • Proteome / isolation & purification
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology

Substances

  • Antibodies
  • Proteome
  • Recombinant Proteins