APOPTOSISKITS - M30‑APOPTOSENSE® ELISA

FAQ

Q1: What is the mode of action for the M30‑Apoptosense^® ELISA?
A: During apoptosis, caspases will cleave various cellular proteins. In epithelial cells, the intermediate filament cytokeratin 18 represents one of the major caspase substrates and is present in abundant quantities. The M30 antibody is a mouse monoclonal of the IgG2b subtype that recognizes a neo‑epitope in the C‑terminal domain of cytokeratin 18 (amino acids 387‑396: CK18Asp396‑NE), which is exposed after cleavage by multiple caspases during apoptosis. Caspases that are capable of cleaving CK 18 to generate this M30 neo‑epitope include in addition to caspase‑3, also caspase‑6, ‑7, and ‑9.

Q2: MCF‑7 cells are known to be defective in caspase‑3 activity. Can I still use the M30‑Apoptosense^® assay?
A: Yes, M30‑Apoptosense^® 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. Apoptosis detection by M30‑Apoptosense^® does not rely on the activity of one particular caspase but can be detected after exposure to agents that may trigger a caspase machinery without the involvement of caspase‑3.

Q3: What can the M30‑Apoptosense^® assay be used for?
A: The M30‑Apoptosense^® is an ELISA for the quantitative detection of the apoptosis‑associated M30 neo‑epitope (CK18Asp396‑NE) in cell extracts, cell culture supernatants, and in human serum or plasma samples given the cells express CK18 (are of epithelial origin). Potential applications e.g.  include HTS screening for apoptotic drug candidates, performing time course kinetics, measuring dose responses after exposure to pro‑apoptotic agents.

Q4: Briefly describe the procedure for the M30‑Apoptosense^® assay.
A: The assay is a solid‑phase, two‑site immunosorbent assay (ELISA). Samples (25 µl) containing caspase‑cleaved CK18 (CK18Asp396‑NE: M30 neo‑epitope) added into the wells bind to an immobilized monoclonal catcher antibody specific to CK18 and are subsequently detected with the CK18Asp396‑NE‑specific M30 monoclonal antibody conjugated to HRP as a tracer. After the formation of the solid phase/antigen/labeled  antibody sandwich, excess unbound tracer is removed by washing. TMB substrate is then added, and the reaction is stopped after a defined incubation period. The absorbance is measured in a microplate reader at 450 nm. By plotting a standard curve from known concentrations vs. measured absorbance, the amount of M30 antigen present in the sample can be calculated. The concentration of the M30 antigen is expressed as units per liter (U/L).

Q5: What is the advantage of using M30‑Apoptosense^® ELISA relative to other methods to quantify apoptotic cell death?
A: M30‑Apoptosense^® is an easy‑to‑use, robust 96‑well format, quantitative screening test specific for apoptosis of  CK18 positive (epithelial) cells. Because the assay 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. This can be combined to use the very same assay to monitor carcinoma cell death in whole organisms by taking serum or plasma samples.

Q6: What is the difference and advantage of the M30‑Apoptosense^® assay compared to an assay that measures e.g. caspase‑3 activity?
A: Both assays measure different processes during apoptosis. The caspase‑3 assay measures enzyme activity (with all the limited specificity problems "peptide caspase"‑substrates have), while the M30‑Apoptosense^® assay does measure the accumulation of a caspase cleavage product (CK18 with the limitation to work only with epithelial cells). Due to the accumulation of the analyte (CK18Asp396‑NE M30 neo‑epitope) over time, M30‑Apoptosense^® is more sensitive and without the need to take into consideration the optimal analytical (often transient) window. It makes this assay convenient and economical when a large number of samples are analyzed. Caspase‑3 activity assays can only be used with samples from cell extracts, while the M30‑Apoptosense^® assay can be used with culture supernatants and human serum or plasma samples.

Q7: I need an apoptosis specific assay that is sensitive and robust.
A: The M30‑Apoptosense^® assay is very sensitive, as it easily detects apoptosis in less than 5,000 carcinoma cells. The stability of the detected CK18Asp396‑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.

Q8: Some methods measure the late stages of apoptosis. What is known for the M30‑Apoptosense^® assay?
A: The M30‑Apoptosense^® assay detects apoptosis early on, as opposed to methods that measure DNA fragmentation.

Q9: Does the M30‑Apoptosense^® assay measure caspase activity? What is the advantage or disadvantage of the M30‑Apoptosense^® assay in this respect?
A: No, the M30‑Apoptosense^® assay does not measure caspase activity. The assay rather measures the amount of accumulated caspase cleavage product (CK18Asp396‑NE M30 neo‑epitope), which provides superior sensitivity and a wider analytical window. If the experimental setting requires the identification of the particular type of caspase activated during apoptosis the M30‑Apoptosense^® assay on its own is not the preferred method. However, given the increasing doubts about the specificity of current commercially available peptide substrates for a particular type of caspase or even for caspases as a subfamily of proteases that become activated during different forms of cell death, the alternative assays may show also their limitations.

Q10: Many established methods for cell death quantification do not distinguish between apoptotic and necrotic cells. What represents the advantage in using the M30‑Apoptosense^® assay?
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 cytokeratin 18 (CK18Asp396‑NE) recognized by the M30‑Apoptosense^® assay, however, is only exposed after cleavage and the M30‑Apoptosense^® assay therefore specifically detects apoptotic cells and not necrotic cells.

Q11: Can the M30‑Apoptosense^® assay be used for all cell types?
A: No, the M30‑Apoptosense^® assay is specific for the CK18Asp396‑NE M30 neo‑epitope, why only CK18‑positive apoptotic cells (of epithelial origin) e.g. breast, lung, prostate, colon, etc can be analyzed. Fibroblasts, lymphocytes, neoronal cells etc. can not be analyzed as these cell types do not express CK18.

Q12: Can the M30‑Apoptosense^® assay also be used for apoptotic CK18‑positive cells from other species?
A: The M30 antibody recognizes caspase‑cleaved cytokeratin 18 (CK18Asp396‑NE M30) from human, rat, mouse and rabbit and can be used for the analysis of apoptosis when the appropriate cell types are used. It is recommended  that the presence of CK18 is confirmed for the individual test cell line. In our experience, the M30‑Apoptosense^® assay shows the highest sensitivity using human samples. Analysis of CK18Asp396‑Ne M30 levels in serum, plasma samples or other body fluids is currently only recommended in the human system.

Q13: Does the M30‑Apoptosense^® assay also detect necrosis?
A: No. Due to the specificity of the M30 antibody to a caspase cleavage product, the M30‑Apoptosense^® assay recognizes apoptotic cells only, not necrotic or viable cells.

Q14: How should samples for the M30‑Apoptosense^® assay be stored?
A: Cell extracts should be frozen at ‑20°C immediately. Serum and plasma samples can be stored for 48 hours at 2 ‑ 8°C. Freezing of samples is recommended if analysis is performed at a later time point.

Q15: What kind of samples can be analyzed by the M30‑Apoptosense^® assay?
A: As the M30‑antigen is also released from epithelial cells, the M30‑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 a combined sample of the former (by direct addition of detergent to the cell culture). The M30‑Apoptosense^® assay can also be used for human serum or plasma. Other body fluids are currently under investigation.

Q16: Can both serum and plasma samples be used for the M30‑Apoptosense^® assay?
A: Yes, the levels (U/L) are similar but not identical. For serial measurements it is recommended to use either plasma or serum for all samples investigated.

Q17: Can some components of the M30‑Apoptosense^® assay 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.

Q18: Does hemolysis in a sample affect the levels of M30‑Apoptosense^® measurements?
A: The M30‑Apoptosense^® assay is NOT sensitive to highly elevated hemoglobin levels, which means that grossly hemolized samples can be assayed although this is not recommended. The results obtained with the M30‑Apoptosense^® assay are also NOT affected by Heparin < 10 U/ml.