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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 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 (CABs), immunohistochemistry has been performed in a similar manner as for HPA-antibodies. These antibodies have also been tested on Western blots. For each commercially available antibody, a link to the antibody provider is given on the "Antigen/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.
All validation scores are classified in three main categories:
- Supportive
- Uncertain
- Not supportive
Validation scores: Immunohistochemistry
- 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.
- Failed: staining pattern not consistent with experimental and/or bioinformatic data.
Antibodies that are validated as Failed are not published.
Validation scores: Immunofluorescence
Since one immunofluorescence validation score is set for each cell line, the scores for the three cell lines are merged into one of the main categories; supportive, uncertain or not supportive, to represent the whole antibody.
- One/multiple localizations supported by literature.
- Multiple localizations partly supported (at least one) by literature.
- One/multiple localizations in cytoplasm (i.e. golgi, mitochondria, ER etc) with literature supporting cytoplasmic localization.
- One/multiple localizations where there is no literature available.
- Not decisive. (One/multiple localizations where the literature is partly supporting and partly conflicting.)
- Annotated weak/cytoplasmic/granular even if supported by literature for cytoplasmic localization.
- No staining.
- Annotated weak/cytoplasmic/granular and there is no literature available.
- Localization not consistent with literature.
Validation scores: Protein array
- Pass with single peak corresponding to interaction only with its own antigen.
- Pass with quality comment low specificity (binding to 1-2 PrESTs >15% and <40%).
- No or weak signal.
- Low specificity (one antigen with >40% signal or more than two antigens with signal >15%).
Antibodies that are validated as 3 or 4 are not published.
Validation scores: Western blot
- Single band corresponding to the predicted size in kDa (+/-20%).
- Band of predicted size in kDa (+/-20%) with additional bands present.
- Single band larger than predicted size in kDa (+20%) but partly supported by predicted transmembrane region, signal peptide or by other available data.
- No bands detected.
- Single band differing more than +/-20% from predicted size in kDa and not supported by predicted transmembrane region, signal peptide or by other available data.
- Weak band of predicted size but with additional bands of higher intensity also present.
- Only bands not corresponding to the predicted size.
- Target too small/large to be analyzed with the present setup.
For antibodies showing not supportive Western blot data the corresponding gel is not shown.
For antibodies supplied through commercial or other academic sources we provide our Western blot and Immunohistochemistry validation scores. For further validation we refer to quality controls provided by respective company.
home | about protein atlas - quality assurance
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