M65® ELISA
(Prod. No. 10020)
M65® ELISA
The M65® ELISA measures soluble CK18 released from dying cells and can be used to assess overall cell death (due to apoptosis and necrosis) of epithelial cells. The M65® ELISA is primarily intended to be used together with the M30-Apoptosense® assay, which specifically measures apoptosis.
As both assays are calibrated against the identical reference the combination of M30-Apoptosense® and M65® ELISA should allow to determine the relative contribution of apoptosis to the total degree of cancer cell death.
Background
Tumor cells die in vivo by apoptosis and necrosis. The mode of cell death depends both on the type of cytotoxic stimulus and the tumor. An apoptotic stimulus may under conditions of deficient cellular ATP generation fail to induce the apoptotic program and cells instead undergo necrosis (Leist et al., 1997). During necrosis, intracellular proteins that are not cleaved by caspases will be released from cells.
Cytokeratin 18 (CK18) is an intracellular protein expressed at high levels by many types of epithelial cells. Most CK18 molecules will form insoluble filaments in the cell, but a pool of soluble CK18 can also be demonstrated (Chou et al., 1993). During cell death, the cellular content of CK18 will be released into the extracellular compartment. Measurements of extracellular soluble CK18 will therefore reflect epithelial cell death "by any cause" (due to apoptosis and necrosis) (Kramer et al., 2004).
CK18 is cleaved by caspases during apoptosis, and caspase-cleaved fragments will be released to the extracellular compartment. The relative proportion of extracellular caspase-cleaved CK18 compared to total CK18 will therefore reflect the relative proportion of apoptosis to total cell death (different "M30:M65 ratios") (Kramer et al., 2004).
The M65® ELISA uses two anti-CK18 mouse monoclonal antibodies of the IgG type and is primarily intended to be used together with the M30-Apoptosense® assay for assessment of tumor cell death using human serum samples. M30-Apoptosense® assay specifically measures a neo-epitope formed by caspase-cleavage of CK18 at Asp396 (CK18Asp396-NE M30 neo-epitope) and will reflect apoptosis of epithelial cells (Hägg et al. 2002; Bivén et al. 2003). The units of the two assays have been calibrated against the identical standard material to allow the calculation of a ratio between caspase-cleaved and total CK18 ("M30:M65 ratio"). Induction of apoptosis in cultured cells will result in release of caspase-cleaved CK18 and in relatively high M30:M65 ratios, whereas induction of necrosis will almost exclusively result in release of CK18 molecules that are not caspase-cleaved and in a low M30:M65 ratio. The M30:M65 ratio will therefore reflect the mode of cell death of epithelial cells.
M65® ELISA is now CE-IVD approved
PEVIVA's M65® ELISA assay has gained CE accreditation as a medical device for in vitro diagnostic use. Updated multi-lingual Instructions for Use are provided with each kit.
