Frequently Asked Questions
M30 CytoDeath™ ELISA
(Updated on January 20, 2009)Q1: What is the intended use of the M30 CytoDeath™ ELISA?
A: The M30 CytoDeath™ ELISA is intended for the quantitative detection of the apoptosis-associated M30 neo-epitope (K18Asp396-NE) in cell extracts or cell culture supernatants. Potential applications include HTS screening for apoptotic drug candidates, performing time course kinetics and measuring dose responses after exposure to pro-apoptotic agents.
Q2: What is the mode of action for the M30 CytoDeath™ ELISA?
A: M30 CytoDeath™ ELISA is a sandwich ELISA based on two mouse monoclonal antibodies, M30 and M6. The M30 antibody recognizes a neo-epitope in the C-terminal domain of keratin 18 (amino acids 387-396: K18Asp396-NE), exposed after cleavage by multiple caspases during apoptosis. The M6 antibody is used as capture antibody in the ELISA. Caspases that are capable of cleaving K18 to generate this M30 neo-epitope include in addition to caspase-3, also caspase-6, -7, and -9.
Q3: What is the advantage of using the M30 CytoDeath™ ELISA relative to other methods to quantify apoptotic cell death?
A: The M30 CytoDeath™ ELISA measures a stable and abundant caspase cleavage product. It offers a very high sensitivity and makes it the method of choice for cost effective drug screening at a functional level, to establish dose-response curves and to establish time course kinetics in cellular systems in therapeutic relevant areas as oncology.
Q4: Can the M30 CytoDeath™ ELISA be used for K18-positive cells from other species than human?
A: The M30 antibody recognizes caspase-cleaved keratin 18 (K18Asp396-NE M30) from human and monkey cells. K18Asp396-NE from mouse, rat, dog and xenopus cells is not detected with sufficient sensitivity. It is recommended that the presence of K18 is confirmed for the individual test cell line.
Q5: What is the difference and advantage of the M30 CytoDeath™ ELISA compared to assays that measure e.g. caspase-3 activity?
A: The caspase-3 assay measures enzyme activity (with specificity problems associated with "peptide caspase"-substrates), while the M30 CytoDeath™ ELISA measures the accumulation of a caspase cleavage product. Due to the accumulation of the analyte (K18Asp396-NE) over time, M30 CytoDeath™ ELISA is more sensitive and does not require consideration of the optimal analytical (often transient) window. This makes this assay convenient and economical when a large number of samples are analyzed. A limitation of the M30 CytoDeath™ ELISA is that it will only work with epithelial cells.
Q6: How specific is the M30 CytoDeath™ ELISA for apoptosis?
A: Some methods, such as TUNEL (labeling of double-stranded DNA breaks) do not distinguish between necrotic and apoptotic cells. The M30 neo-epitope on keratin 18 is only exposed after caspase cleavage occuring during apoptosis.
Q7: Can the M30 CytoDeath™ ELISA be used for all cell types?
A: No, the M30 CytoDeath™ ELISA can only be used for K18-positive cells (of epithelial origin) e.g. breast, lung, prostate, colon etc. Fibroblasts, lymphocytes, neuronal cells etc. can not be analyzed as these cell types do not express K18.
Q8: Does the M30 CytoDeath™ ELISA also detect necrosis?
A: No. Due to the specificity of the M30 antibody to a caspase cleavage product, the M30 CytoDeath™ ELISA recognizes apoptotic cells only, not necrotic or viable cells.
Q9: How should samples for the M30 CytoDeath™ ELISA be stored?
A: Cell extracts should be frozen at -20°C immediately, if not used within 4 hours.
Q10: What kind of samples can be analyzed by the M30 CytoDeath™ ELISA?
A: As the caspase-cleaved product is released from cells, the antigen is present in the cell culture supernatant as well as in cytosolic cell extracts. Both types of samples can therefore be analyzed individually, or as a combined sample (by direct addition of detergent to the cell culture).
Q11: Is the M30 CytoDeath™ ELISA sensitive and robust?
A: The assay is very sensitive, as it easily detects apoptosis in less than 5 000 carcinoma cells. The stability of the detected K18Asp396-NE M30 neo-epitope makes the assay very robust and reproducible. The established HRP-TMB ELISA method is very reproducible compared to e.g. caspase activity assays that require an optimal reaction buffer, and are sensitive to changes in temperature and substrate concentration.
Q12: Briefly describe the procedure for the M30 CytoDeath™ ELISA.
A: Serum samples (25 µl) containing caspase-cleaved K18 (K18Asp396-NE: M30 neo-epitope) bind to an immobilized monoclonal capture antibody specific to K18. HRP-conjugated M30 antibody is added and the plates are incubated for 4 hours. Excess unbound tracer is removed by washing and TMB substrate is added. The reaction is stopped after a defined incubation period and the absorbance is measured in a microplate reader at 450 nm.
A: Yes, M30 CytoDeath™ ELISA can be used in cells lacking active caspase-3 as also other caspases, but no other proteases can generate the M30 neo-epitope. Apoptotic MCF-7 cells generate M30-activity due to activity of those other caspases.
Q14: Some methods measure the late stages of apoptosis. What is
known for the M30 CytoDeath™ ELISA?
A: The M30 CytoDeath™ ELISA detects apoptosis early on, as
opposed to methods that measure DNA fragmentation.
Q15: Can some components of the M30 CytoDeath™ ELISA from
different lots be mixed or combined?
A: No, it is strongly advised to use the components of the same lot
provided with each kit only as these may affect the consistency of the
results obtained.
