The Swedish Human Protein Atlas project, funded by the (non-profit) Knut and Alice Wallenberg Foundation, invites submission of antibodies from both academic and commercial sources to be included in the human protein atlas (www.proteinatlas.org). All antibodies will be validated by the Human Protein Atlas project by a standard procedure and antibodies that are accepted will be use in the tissue-profiling program to generate high-resolution immunohistochemistry images representing a wide spectrum of normal tissues and cancer types. During the pilot project phase, the validation will be done free of charge. For each antibody, 576 spots of human tissue from 360 different individuals are immunostained and manually annotated. Academic collaborators will have access to all biological data at least 6 months before the images will be published as part of the Human Protein Atlas. Commercial vendors will receive a direct web-link from the antibody information page on the Protein Atlas to a web-link decided by the antibody provider.
The collaborator is asked to fill-in an excel-sheet submission form with information, such as:
- Gene/Protein identifier of target (Ensembl identifier)
- Description of target used for the antibody generation
- Description of type of antibody (pAb, mAb of recAb)
- Results from quality assurance (preferably a web-link but otherwise pdf-file)
Information about the antigen is also requested in the submission form. It is preferable (but optional) to give the size of the antigen in number of amino acids. In the comment field one can also add actual peptide sequence and information about purity check, sequence verification etc (optional).
Submissions could range from a single antibody to several hundreds of antibodies. Use one line in the excel sheet per antibody. Send the submission form (excel) to: firstname.lastname@example.org. Submission instructions can be found here.
Not more than two antibodies to the same target protein will normally be pursued. Priority will thus be given to new targets not present (or only represented by one antibody) in the Human Protein Atlas portal or in the Human Protein Atlas pipeline. Priority will be given to antibodies with well-documented and relevant quality assurance. A documentation of the validation should be submitted preferably through a web-link (product data sheet and/or reference) or alternatively as a pdf-file (with results, reference and/or product data sheet). Antibodies should be validated by at least one of the following two methods:
- Western blot (with relevant human tissue extracts and/or cells)
- Immunohistochemistry (IHC with relevant human tissue and/or cells)
Examples of other suitable validation assays are:
- Antigen-based assays (ELISA, protein arrays or similar protein assay)
- Adsorption test (in combination with IHC)
- Flow sorting
- Immunocapture results ("pull-down" followed by suitable analysis)
For submissions that are approved, the collaborator will be asked to send in a vial (free of charge) of each antibody approved. A minimum amount of 50 µg or equivalent will be needed for each antibody.
Use of the antibodies by the HPA program
Each antibody is tested according to HPA-standard quality assurance procedures, i.e., on Western blots and on a special designed tissue microarray (TMA) initially using two antibody-dilutions and heat epitope retrieval in citrate buffer. In selected cases, other pre-treatment, EDTA buffer or enzyme based antigen retrieval methods will be tried. The number of test rounds for each antibody is restricted to maximum three. Each antibody approved by HPA will subsequently be used for immunostaining of eight TMAs representing 576 tissue spots.
Flow of information
The antibody provider (academic or commercial) will obtain the results of the quality assurance. Confirmation of successful tissue profiling (including annotation) will be sent to the collaborator. Information about non-approved antibodies will only be communicated to the antibody provider (and will not be published). For academic collaborators, the data will be stored at least six months on the internal web atlas before transfer to the publicly available Protein Atlas (www.proteinatlas.org). Each approved antibody on the public database will be directly linked from the Protein Atlas to the collaborators web-home page.
The cost for the profiling (Western blot analysis, immunohistochemistry, scanning and annotation) will (during the pilot project) be covered by the Human Protein Atlas project (Wallenberg Foundation).
Release of data
Academic collaborators, can ask that the release of the data on the public Human Protein Atlas portal should be delayed further (beyond six months) if publication(s) based on the results from the protein profiling are being prepared. In this case, the release of the images can be delayed (pending decision by the HPA priority committee) normally not longer than one year.