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PathHunter® Technology for Cell-Based Assays

DiscoveRx has exploited the versatile features of enzyme fragment complementation (EFC) technology to create cell-based assays for a vast number of proteins.  PathHunter assays are designed using two inactive fragments of β-galactosidase (β-gal), which combine to create an active enzyme.

Learn about EFC

Four Applications of EFC for Biologics Development

 

Receptor Activation

Receptor Internalization

receptor activation Receptor Internalization
 

Receptor Dimerization

Receptor Signaling

Receptor Signaling
 

EFC technology can be used to study multiple cell surface receptors and their ligands  By exploiting the natural biology of various receptors, PathHunter EFC technology can be used to create simple, cell-based chemiluminescent assays. (A) By tagging the receptor and the activation-based intracellular response protein, we can create a specific assay for an activated receptor, e.g. β-Arrestin recruitment to an activated GPCR. (B) By tagging the early endosome and the receptor, we can follow receptor internalization for GPCRs, RTKs and other receptors. (C) By tagging various receptors we can monitor the formation of receptor heterodimers and homodimers. (D) By tagging cellular pathway proteins, signaling events downstream of receptor activation including cAMP accumulation, protein translocation or protein degradation can be measured.


Applications of PathHunter Assays

 

Anti-Receptor Antibody

    

Anti-Ligand Antibody

Functional anti-ErbB3 antibodies (panel A) show a dose-dependent inhibition of signal in the ErbB2-ErbB3 assay, which detects the recruitment of SH2-domain protein upon activation of the receptor.  Anti-ligand antibody (panel B) against GLP1 demonstrates dose-dependent inhibition in the GLP1R assay, which detects the recruitment of β-arrestin upon activation of the GPCR.

Receptor Internalization

    

Dimerization Inhibitors

A receptor internalization assay (panel C) with an agonist for OPRD1, a GPCR opioid receptor, detects internalization of the GPCR to the late endosome. Anti-receptor antibody (panel D) against TGF βR2 demonstrates dose dependent inhibition in the TGF βR1/R2 dimerization assay, which detects the dimerization of the two receptors when bound by their ligand.