Human Protein Atlas

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Quality assurance

The usefulness of antibodies in different assays is dependent on both sensitivity and specificity of epitope binding. The quality of antibodies in the database is monitored through a number of different quality assurance steps. Below is a list of measures taken to ensure that the quality of produced and utilized PrEST antibodies is acceptable. All PrEST antibodies must pass steps 1-3 in order to be used for immunohistochemistry. Steps 4-5 provide a basis for an evaluation and scoring of antibody validity. All antibodies that provide a reasonable pattern of immunoreactivity are added to the protein atlas. Feed-back from the research community is appreciated and needed for continuous curation of data.

Quality assurance steps for PrEST antibodies generated within HPA:

Plasmid inserts are sequenced to assure that the correct PrEST sequence is cloned.
Size of resulting recombinant protein (including the specific PrEST) is analyzed using mass spectrometry to assure that the correct antigen has been produced and purified.
To control for cross-reactivity, affinity purified antibodies are tested for sensitivity and specificity on protein arrays consisting of glass slides with spotted PrEST fragments.
Antibody specificity is analyzed using Western blot in a standardized setup. Total protein lysates from a limited number of cell lines and tissues (liver and tonsil) and cell lines (RT-4 and U-251MG) are used to evaluate the antibody target binding in a Western blot setting.
Immunohistochemical staining of normal and cancer tissue is examined by trained pathologists to assure plausible immunohistochemical staining properties.

For commercially available antibodies, immunohistochemistry has been performed according to the manufacturers instructions when such have been available. Immunostaining protocols have been tested to obtain reasonable results with suggested positive control. These antibodies have also been tested on Western blots. For each commercially available antibody, a link to a providing antibody company is given on the "Antibody info" page.

The validation score indicates how well the quality assurance data supports the specificity of the antibody towards the expected human target protein. The HPA antibodies were classified in four main categories based on the immunohistochemical staining pattern, Western blot analysis and presence/absence of literature and bioinformatic data.

High: two independent antibodies targeting one protein yielding similar staining patterns. Staining pattern consistent with experimental and/or bioinformatic data.
Medium: staining pattern consistent with experimental and/or bioinformatic data.
Low: staining pattern partly consistent with experimental and/or bioinformatic data.
Very low: no experimental and/or bioinformatic data available, or staining pattern not consistent with experimental and/or bioinformatic data.

For antibodies supplied through commercial or other academic sources we refer to internal quality controls provided by respective company.

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