Frequently Asked Questions
M30-Apoptosense® ELISA
(Updated on January 20, 2009)Q1: What is the intended use of the M30-Apoptosense® assay?
A: The M30-Apoptosense® ELISA is intended for the quantitative detection of
the apoptosis-associated M30 neo-epitope (CK18-Asp396-NE) in blood samples.
Q2: What is the mode of action of the M30-Apoptosense® ELISA?
A: M30-Apoptosense® assay is a sandwich ELISA based on two mouse monoclonal antibodies, M30 and M5. The M30 antibody recognizes a neo-epitope in the C-terminal domain of cytokeratin 18 (amino acids 387-396: CK18Asp396-NE), exposed after cleavage by multiple caspases during apoptosis. The M5 antibody is used as capture antibody in the ELISA.
Q3: What is the species specificity of the M30-Apoptosense® assay?
A: The M30 antibody recognizes caspase-cleaved cytokeratin 18 (CK18-Asp396-NE M30) from human, monkey, cow and pig origin. CK18-Asp396-NE from mouse, rat, dog or xenopus cells is not effectively detected.
Q4: 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 a particular project or series of measurements, it is recommended to use either plasma or serum for all samples investigated.
Q5: How should serum or plasma samples for the M30-Apoptosense® assay be stored?
A: Serum and plasma samples should be stored on ice or at +4 before freezing at -80oC.
Q6: Is the M30- Apoptosis® assay sensitive and robust?
A: The M30-Apoptosense® assay is very sensitive. The stability of the detected CK18Asp396-NE M30 neo-epitope makes the assay very robust and reproducible.
Q7: Briefly describe the procedure for the M30-Apoptosense® assay.
A: Serum samples (25 µl) containing caspase-cleaved CK18 (CK18Asp396-NE:
M30 neo-epitope) bind to an immobilized monoclonal capture antibody specific to CK18. 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.
Q8: 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.
Q9: Does hemolysis in a sample interfere with M30-Apoptosense® measurements?
A: The M30-Apoptosense® assay is NOT sensitive to highly elevated hemoglobin levels, which means that haemolized 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.
Q10: Does the M30-Apoptosense® assay measure caspase activity?
A: No, the M30-Apoptosense® assay does not measure caspase activity. The assay measures the amount of accumulated caspase cleavage product, which provides superior sensitivity and a wider analytical window.
