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.
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