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
SGC Binder Name Target HGNC Name NCBI Entrez Gene ID UniProt ID Antigen domain Target Annotation Binder generation lab Validated binder format Commercial product available Approximate KD (nM) Full scFv Sequence (VH-linker-VL)
AC-FARSA-1 FARSA 2193 Q9Y285 A2-A508 of 508, Full-length phenylalanyl-tRNA synthetase, alpha subunit DDD scFv   8 FASTA
AC-GLE1-4 GLE1 2733 Q53GS7 Q383-S698 of 698 Nucleoporin GLE1 DDD scFv   1 FASTA
AC-PFN1-4 PFN1 5216 P07737 M1-Y140 of 140, Full-length Profilin-1 DDD scFv   18 FASTA
AC-ANXA11-1 ANXA11 311 P50995 1-505 of 505, Full-length Annexin A11 DDD scFv   50 FASTA
J-HARS-56 HARS 3035 P12081 1-509 of 509, Full-length Histidyl-tRNA synthetase DDD scFv   1 FASTA
O-Parkin-10 PARK2 5071 O60260 M1-V465, Full-length (R. Norvegicus) E3 ubiquitin-protein ligase parkin DDD scFv   2 FASTA
AC-EPRS-5 EPRS 2058 P07814 A2-G196 of 1512 Bifunctional glutamate/proline--tRNA ligase DDD scFv   3 FASTA
DARPIN-A10 MAP1LC3A 84557 Q9H492 LC3A Microtubule Associated Protein 1 Light Chain 3 Alpha VD DARPIN   5  
DARPIN-A6 TP63 8626 Q9H3D4 Sterile alpha motif domain Tumor protein 63 VD DARPIN   25  
DARPIN-6E3 TP63 8626 Q9H3D4 Tetramerization domain Tumor protein 63 VD DARPIN   40