Antibodies

Recombinant antibodies & Biological Probes within the EUbOPEN project

As part of work package 4 of the EUbOPEN project, recombinant antibodies are being generated to selected target proteins to be used as research tools. These antibodies have been validated to recognize their endogenous targets in a complex mixture and can thus be used for cell-based assays like immunoprecipitation (IP) and immunofluorescence (IF). A subset of the antibodies are declared biological probes and can be used for target validation. These are targeting proteins present in the extracellular matrix and must be shown to have an effect on live cells in an assay format or to have a diagnostic use by detection of specific proteins in biological samples. The best antibody for each target protein will be made available through disclosing the sequence here.

Antibody validation method

Selected antibody candidates are first validated for their binding against their antigen using various immune-based assays in 96-well format. Binders that pass this initial validation are then subjected to cell-based validation, testing whether they can bind their target antigens produced endogenously in a human cell. This is done via immunoprecipitation followed by mass spectrometry, or IP-MS for short. The strength of this method is in its ability to detect not only the target protein that the antibody pulls down, but also its protein complex partners, and potential cross-reactivity targets. Compared to other traditional methods like western blot where you see only a band on a gel, IP-MS also provides the identity of each protein in the immunoprecipitate.

Antibody validation criteria

• Binds its target antigen with an affinity of at least 20 nM
• Can immunoprecipitate its endogenous target protein from HEK293 cell lysate (or other relevant cell line or biological sample)
• Specific in IP-MS, or at least that any cross-reactivity is known

Additional for biological probes

• Measurable modulation of function in relevant cell assays or potential diagnostic/biomarker use