The Johns Hopkins University (JHU) / CDI Laboratories, Inc. (CDI) production center exploits a unique human proteome technology, the HuProt array with almost 20K spotted protein isoforms corresponding to ~15K unique human genes, in a pipeline to generate monospecific monoclonal antibodies (mAbs) against human transcription factors. Protein antigens have been provided as domains supplied the Rutgers production center and within the JHU/CDI center as full-length proteins from a comprehensive collection of human full-length clones.
The antigens are used for mouse immunizations to generate hybridoma clones, which are then selected for IgG secretion. Individual clones are harvested and expanded, and then pass through primary and secondary validations. Unlike conventional pipelines that only assess affinity, the JHU/CDI primary validation uses mini-chips of spotted protein targets followed by binding assays with the full HuProt to ensure specificity of binding in addition to affinity. Monospecific mAbs that pass a primary validation (HuProt, OIRD, or Octet) must then pass a secondary validation, including immunoprecipitation (IP) and immunoblotting/western blotting (WB), to be considered passing reagents. Full criteria for passing individual mAbs for display on this portal and distribution are available for download here. Other secondary validations for additional applications include ChIP-Seq (ENCODE quality standards), ELISA, immunoprecipitation mass spectrometry (IP-MS), surface plasmon resonance (SPR), and tissue immunohistochemistry (IHC).
The mAbs that pass primary and secondary validation are available at low cost from the Developmental Studies Hybridoma Bank (DSHB) and also directly from CDI. Protocols for standard applications are available for download from the PCRP Data Portal protocols page.