Application

For research use only. Not for use in diagnostic procedures.Specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates.

Cell Death Detection ELISA has been used to measure Caspase-3 activity, intracellular reactive oxygen species (ROS) and cell apoptosis.

General description

The Cell Death Detection ELISA serves as photometric enzyme immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induced cell death. This assay is based on the quantitative sandwich-enzyme immunoassay-principle using mouse monoclonal antibodies directed against DNA and histones, respectively. The antibodies used are not species specific.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Packaging

1 kit containing 9 components.

Preparation Note

Working solution: Coating solutionPredilute 1 ml Coating buffer conc. with 9 ml double dist. water. Shortly before use, dilute 1 ml anti-histone antibody (reconstituted) with 9 ml Coating buffer.Washing solutionWarm the Washing buffer concentrate to 15 to 25 °C and dilute 40 ml in 360 ml double distilled water. Mix thoroughly.Sample solutionPreparation of the sample solution depends on the cell system used and the extent of cell death.Example: Dilute 25 µl of sample in 225 µl Incubation buffer.Conjugate solutionDilute 1 ml Anti-DNA-POD (reconstituted) with 9 ml Incubation buffer.Substrate solutionDepending on the number of samples tested, dissolve 1, 2, or 3 tablets in 5, 10, or 15 ml Substrate buffer.Allow to come to 15 to 25 °C before use.Note: The ABTS solution is light sensitive over extended periods of time.Storage conditions (working solution): Anti-histone2 to 8 °C for 2 monthsAnti-DNA-POD2 to 8 °C for 2 monthsCoating solutionPrepare immediately before use!Washing solution2 to 8 °C for 2 monthsSample solution2 to 8 °C for 2 monthsConjugate solutionPrepare immediately before use!Substrate solution2 to 8 °C for 1 month, protected from light.

Reconstitution

Anti-histoneReconstitute the lyophilizate in 1 ml double-distilled water for 10 minutes.Mix thoroughly.Anti-DNA-PODReconstitute the lyophilizate in 1 ml double-distilled water for 10 minutes. Mix thoroughly.

Specificity

Anti-histone antibody reacts with the histones H1, H2A, H2B, H3, and H4 of various species (e.g., human, mouse, rat, hamster, cow, opossum, and Xenopus). Anti-DNA-POD antibody binds to ss- and dsDNA. Therefore, the ELISA allows the detection of mono- and oligonucleosomes from various species, and may be applied to measure apoptotic cell death in many different cell systems.