AffinityImmuno Products

Introduction

GSDME (also known as DFNA5) was first identified through its link to inherited hearing loss, but it is now understood chiefly as an executioner of a lytic, inflammatory form of cell death called pyroptosis. The protein sits inertly in cells until it is cut, either by caspase-3 during what would otherwise be a quiet apoptotic death or by granzyme B delivered from a cytotoxic natural killer or T cell, and this cleavage frees an N-terminal fragment that seeks out the inner face of the plasma membrane. There, the fragment inserts itself and assembles into large ring-shaped pores roughly ten to fifteen nanometers across, rupturing the cell and releasing mature inflammatory cytokines such as IL-1 beta in the process, effectively converting a silent death into an inflammatory one. This switch can work to a tumor’s disadvantage, since granzyme-driven pyroptosis of cancer cells helps recruit and activate anti-tumor immune responses, giving GSDME a tumor-suppressive character even though its original discovery had nothing to do with cancer biology.

Because granzyme-driven cleavage of GSDME converts tumor cell death into an inflammatory, immune-activating process, this assay is relevant to the fast-growing pyroptosis-immunotherapy research field studying how to harness programmed cell death pathways to boost anti-tumor immune responses.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format to measure Human Gasdermin E (GSDME). Microtiter plate wells are pre-coated with a GSDME-specific capture antibody that binds the target analyte present in standards or samples. A biotin-conjugated detection antibody, also specific to Human GSDME, is then added and binds a second epitope on the captured analyte. Avidin conjugated to horseradish peroxidase (HRP) is subsequently introduced and binds the biotin label. Upon addition of TMB substrate solution, a colorimetric signal develops exclusively in wells containing GSDME, the biotin-conjugated detection antibody, and avidin-HRP. The enzymatic reaction is stopped by the addition of sulphuric acid solution, and absorbance is read at 450 nm (± 10 nm). GSDME concentrations in unknown samples are determined by referencing their optical density values against a standard curve.