The M65 EpiDeath® ELISA kit measures the concentration of soluble CK18 (K18) in cell culture supernatants, serum or plasma for research studies. CK18 levels reflect the amount of total epithelial cell death, regardless of the cause of death.
Why are there 2 M65 ELISAs?
The M65® ELISA was introduced in 2004 and intended for the determination of total cell death in research of epithelial cells. A combined use of M65® ELISA and M30 Apoptosense® ELISA facilitates the determination of cell death mode (apoptosis or necrosis).
The M65 EpiDeath® ELISA is based on the same monoclonal antibodies as the M65® ELISA, except mAb M5 is used as the capture antibody (mAb M6 is used as capture antibody in the M65® ELISA). Reaction buffers have also been slightly modified This CK18 kit is manufactured differently and thus measures the (complex bound) CK18 epitopes differently than the M65® ELISA, which leads to a better discrimination between low or normal and high levels. It has a broad range as well.
The M65 EpiDeath ELISA is recommended whenever a customer wants to identify cell death with high sensitivity and specificity in research. The M65 ELISA is the better choice for customers intending to determine the cell death mode when using in combination with the M30 Apoptosense ELISA, especially when using a mathematical approach to describe the distribution between apoptosis and necrosis.
- Only method available to specifically measure epithelial apoptosis and necrosis in the blood
- Specific quantification measurement tool for apoptosis and necrosis in CK18 positive cells
- Sandwich ELISA in a 96 well plate in a convenient ready-to-use format
- Easy to perform, only a minimum of pipetting steps required
- It can be split up for use at several occasions
- CK18 is present in simple epithelial cells only, thus increasing specificity of measurement in epithelial-based models
- Remarkable stability of the CK18 protein complexes in the circulation, thus providing stable storage of serum/plasma samples and allowing for multiple freeze thaw cycles.
- Minimal day-to-day fluctuations in healthy subjects
- Suitable to use together with M30 Apoptosense® ELISA for quantification of apoptosis, necrosis and total cell death
Reagents, Packaging, Storage, and Stability
- M5 Coated Microstrips: One microplate, 12 strips with 8 wells each, 96 dry wells in total. The wells are coated with mouse monoclonal K18 antibody M5. The microplate is sealed in an aluminium bag, which contains a desiccating device. If not all the strips are used, reseal the bag and keep the desiccating device inside. Ready for use!
- M65 EpiDeath Conjugate: Concentrate (24 × conc.). One vial containing 0.4 mL of mouse monoclonal M6 antibody (anti-K18) conjugated with horseradish peroxidase (HRP) in a phosphate buffer with protein stabilizers. Preservative added. Should be diluted with M65 EpiDeath Conjugate Dilution Buffer. Note! Do not expose to light!
- M65 EpiDeath Conjugate Dilution Buffer: One vial containing 11 mL of phosphate buffer with protein stabilizers for dilution of the M65 EpiDeath Conjugate. Preservative added. Blue colored.
- M65 EpiDeath Standard A–H: Standard A containing 2 mL of phosphate buffer with fetal calf serum (FCS). Standard B–H, 0.5 mL each, containing standard material in phosphate buffer with FCS. The values of Standard A–H are 0, 200, 400, 800, 1 200, 2 000, 3 000, and 5 000 U/L, respectively. Preservative added. Yellow colored. Ready for use! Standard A can be used for dilutions of samples > 5 000 U/L.
- M65 EpiDeath Control Low & High: Two vials containing 0.5 mL of reactive components in phosphate buffer with FCS. The values of M65 EpiDeath Control Low and M65 EpiDeath Control High are stated on the respective vials. Preservative added. Yellow colored. Ready for use!
- Wash Tablet: One tablet for 500 mL of prepared wash solution. Dissolve the Wash Tablet in 500 mL of fresh deionised water.
- TMB Substrate: One bottle containing 22 mL of TMB (3,3’,5,5’-Tetramethylbenzidine) Solution. Note! Do not expose to light! Ready for use!
- Stop Solution: One vial containing 7 mL of 1.0 M sulphuric acid. Ready for use!
- Sealing Tape: One (1) sheet.
The M65 EpiDeath® ELISA is a solid-phase sandwich enzyme immunoassay. Standards, controls and samples react with a solid phase capture antibody M5 directed against K18 and the HRP- (Horseradish Peroxidase) conjugated M6 antibody directed against a different epitope of K18. Unbound conjugate is removed by a washing step. TMB Substrate is added. The color development is stopped and the absorbance is read. The resulting color is directly proportional to the concentration of the analyte. By plotting a standard curve from known concentrations versus measured absorbance, the amount of antigen in the sample can be calculated. The concentration of the antigen is expressed as units per liter (U/L).
Cells die by apoptosis or necrosis. The mode of cell death depends both on the type of stimulus (e. g. the cytotoxic agent and tumour upon chemotherapy, or the mechanism of injury upon liver diseases) and the type of tissue (e. g. the tumour). The combination of the M30 Apoptosense ELISA and the M65 EpiDeath ELISA quantifies not only the amount of cell death, but also the cell death mode (apoptosis or necrosis) in blood samples (serum or plasma) or cell supernatants.
Cytotoxic agents used in oncology try to overcome the disruption of the apoptotic process and stimulate apoptosis. Dependent on the type of tumour and the cytotoxic agent, necrosis can be triggered instead. An apoptotic stimulus may under conditions of deficient cellular ATP generation fail to induce the apoptotic program and cells instead undergo necrosis.
When researching the liver, injury may lead to cell death of hepatocytes – either by necrosis or apoptosis.
Keratin 18 (K18) is an intracellular protein expressed at high levels by many types of epithelial cells. Most K18 molecules will form insoluble filaments in the cell, but a pool of soluble K18 can also be demonstrated. During cell death, the cellular content of K18 will be released into the extracellular compartment. Research measurements of extracellular soluble K18 will therefore reflect epithelial cell death “by any cause” (due to apoptosis and necrosis).
K18 is cleaved by caspases during apoptosis, and caspase-cleaved fragments will be released to the extracellular compartment. The concentration of extracellular caspase-cleaved K18 (ccK18) therefore reflects the amount of apoptosis. Furthermore, the M30:M65 ratio is an indication of the proportion of apoptosis compared to total cell death.
The M65 EpiDeath® ELISA uses two anti-K18 mouse monoclonal antibodies of the IgG type and is intended for assessment of epithelial cell death using human serum or plasma research samples. The M30 Apoptosense® ELISA specifically measures a neo-epitope formed by caspase-cleavage of K18 at Asp396 (K18Asp396-NE or M30 neo-epitope) and will reflect apoptosis of epithelial cells. 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 K18 (“M30:M65 ratio”). Induction of apoptosis in cultured cells will result in release of caspase-cleaved K18 and in relatively high M30:M65 ratios, whereas induction of necrosis will almost exclusively result in release of K18 molecules that are not caspase-cleaved and in a low M30:M65 ratio.