Background

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor of the CD28 Ig superfamily, mainly on activated T and B cells, NK cells, and myeloid cells. It helps maintain immune tolerance by suppressing T cell activity and preventing autoimmunity. Tumors exploit PD-1 to evade immune attack by binding its ligands PD-L1/PD-L2, inhibiting TCR signaling and causing T cell exhaustion. This mechanism led to immune checkpoint inhibitors (ICIs) that block PD-1/PD-L1, restoring T cell function and enhance tumor destruction.

Eurofins DiscoverX’s PD-1 Bioassay Kit (SHP1 Signaling) models this MOA in a simple, homogenous, miniaturizable format, suitable for implementation in characterization, QC lot release, stability, and detection of neutralizing antibodies.

Product Highlights
  • Designed for interrogating PD1/PD-L1 signaling analysis
  • Detects and measures SHP1 recruitment to activated PD-1 receptor
  • Ready-to-use, cell-based assay format
  • Suitable for rank ordering, potency measurement, stability studies and neutralizing antibody detection
  • Applicable across various stages from characterization and QC lot-release

Assay Principle

In PathHunter PD-1 Signaling Assay, a full-length PD-1 receptor was co-expressed with SHP1 in Jurkat cells. PD-1 receptor was tagged with an Enzyme Donor (ED, shown in orange) fragment of β-galactosidase (β-gal) at the C-terminus (cytoplasmic tail), and SHP1 tagged with Enzyme Acceptor (EA, shown in blue) of β-gal. Ligand engagement through PD-L1 with PD-1 results in SHP recruitment, bringing the EA and ED fragments together. ED and EA are inactive β-gal fragments, but when brought together, their complementation (termed as enzyme fragment complementation or EFC) forms an active β-gal enzyme that hydrolyzes a substrate to generate a chemiluminescent signal. However, when a therapeutic agent blocks the interaction between PD-1 and its ligand, SHP recruitment does not occur, resulting in loss of EFC signal.

SHP1 Signaling Assay - Assay Principle