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Creation of an antibody resource

An important issue in the initial phases of the HPA effort was the choice of strategy to create a resource for a genome-wide coverage of antibodies to the human proteins. Monoclonal antibodies (and recombinant antibodies) are characterized by having a single binding site (epitope) and importantly these reagents have the advantage to be a renewable resource through culturing of hybridoma cells (or recombinant expression). Polyclonal antibodies on the other hand have several binding sites (multiple epitopes). The main advantage with this is that they are less vulnerable when the structure of the target protein is disrupted (denaturated) and thus less dependent on sample handling by individual researchers. However, it is important to point out that polyclonal serum has the disadvantage that a large fraction of generated antibodies in the polyclonal serum recognize unrelated proteins, a fact that can often cause undesired cross-reactivity. A new concept was developed to avoid this issues. Polyclonal serum was generated and the serum was subsequently affinity-purified using the antigen as ligand to create “antigen-purified antibodies”, also called “mono-specific antibodies”. In this way, only antibodies that binds to the intended target remains after affinity purification, but the antibodies generated in this manner still bind to multiple epitopes on the target protein. More than 55,000 antigen-purified antibodies were generated “in-house” using an automated chromatography systems for specific affinity purification using the corresponding purified recombinant antigens. The antigen-purified antibodies were validated using a a comprehensive scheme involving protein micro arrays, immunohistochemistry and Western blot analysis and more than 21,000 of the antibodies passed the analysis and these were used for tissue profiling using tissue micro arrays.

All antibodies generated within the HPA effort have been made available to the research community through the Atlas Antibodies portal (www.atlasantibodies.com). (All antibodies have been validated by tissue profiling in more than 700 tissue samples and annotated by a certified pathologist. Several international efforts have been initiated to provide antibodies to the research community, and HPA has participated in many of these efforts, including the European Union framework program ProteomeBinders and the U.S. National Institutes of Health Protein Capture Reagents Program.

Key publication

  • Nilsson P et al., Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling. Proteomics. (2005)
    PubMed: 16237735 DOI: 10.1002/pmic.200500072

Other selected publications

  • Taussig MJ et al., ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.  Nature Methods (2007)
    PubMed: 17195019 DOI: 10.1038/nmeth0107-13

  • Stoevesandt O et al., European and international collaboration in affinity proteomics. N Biotechnol. (2012)
    PubMed: 22682155 DOI: 10.1016/j.nbt.2012.05.003

  • Gloriam DE et al., A community standard format for the representation of protein affinity reagents. Mol Cell Proteomics. (2010)
    PubMed: 19674966 DOI: 10.1074/mcp.M900185-MCP200

Figure legend: The accumulated number of unique antibodies used to perform the protein profiling published in the HPA each year, starting in 2005.

Key facts

  • More than 55,000 antibodies have been generated in-house, and approximately 21,000 have passed the stringent annotation of the HPA program for use in immunohistochemistry
  • More than 10 million tissue and cell images have been generated using this antibody resource
  • The validation data for each antibody is shown on the “antibodies and validation” page of each gene
  • All antibodies used in the HPA program are available to the scientific community through the Atlas Antibodies portal (www.atlasantibodies.com)