Cell Death Detection ELISA PLUS 细胞凋亡

• One-step ELISA
• High sensitivity: (5 x 10² cells/ml)
• Fast performance: (3 - 4 hours)
• Positive control included
• No prelabeling of cells necessary
• Nonradioactive assay system
• No species restriction
• Easy handling
• Low background
• Suppressed human anti-mouse factor
• Function-tested

Assay time:
3 - 4 hours
Negative control:
Depending on cell culture conditions, each exponentially growing permanent cell culture contains a certain amount of dead cells (typically approximately 3 - 8%). In the immunoassay, these inherent dead cells in the untreated sample (without a cell-death-inducing agent) will cause a certain absorbance value (negative control).
Positive control:
A DNA-histone complex serves as a positive control.
Sample material:
Cytoplasmic fractions (lysates) of cell lines, cells ex vivo, cell culture supernatants, and serum or plasma
Sensitivity:
The exact detection limit of dying/dead cells in a particular sample strongly depends on the kinetics of cell death, the cytotoxic agent used, and the amount of affected cells in the total cell population. Using U937/camptothecin (CAM) as a cellular model system for cell death, the immunoassay allows the specific detection of mono- and oligonucleosomes in the cytoplasmic fraction of 125 cell equivalents/well.
Specificity:
Anti-histone reacts with the histones H1, H2A, H2B, H3, and H4 of various species (e.g., human, mouse, rat, hamster, cow, opossum, Xenopus). Anti-DNA binds to single- and double-stranded DNA. 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.

Two distinct forms of eukaryotic cell death can be classified by morphological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased ion permeability of the plasma membrane; the cells swell and the plasma membrane ruptures within minutes (osmotic lysis). Apoptosis is characterized by membrane blebbing (zeiosis), condensation of cytoplasm, and the activation of an endogenous endonuclease. This Ca²⁺- and Mg²⁺-dependent nuclease cleaves double-stranded DNA at the most accessible internucleosomal linker region, generating mono- and oligonucleosomes. In contrast, the DNA of the nucleosomes is tightly complexed with the core histones H2A, H2B, H3, and H4, and is thus protected from cleavage by the endonuclease. The yielded DNA fragments are discrete multiples of an 180-bp subunit, detected as a “DNA ladder” on agarose gels after extraction and separation of the fragmented DNA. The enrichment of mono- and oligonucleosomes in the cytoplasm of the apoptotic cell is due to the fact that DNA degradation occurs several hours before plasma membrane breakdown.

1. Anti-histone-biotin (clone H11-4)
2. Anti-DNA-POD (clone MCA-33)
3. Positive Control
4. Incubation Buffer
5. Lysis Buffer
6. Substrate Buffer
7. ABTS Substrate Tablet
8. ABTS Stop Solution
9. Microplate (streptavidin-coated)
10. Adhesive Cover Foils

The assay is based on the quantitative “sandwich enzyme immunoassay” principle using mouse monoclonal antibodies directed against DNA and histones. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. The samples are placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase. During the incubation interval, nucleosomes will be captured via their histone component by the anti-histone-biotin antibody, while binding to the streptavidin-coated microplate. Simultaneously, anti-DNA-peroxidase binds to the DNA part of the nucleosomes. After removal of the unbound antibodies, the amount of peroxidase retained in the immunocomplex is photometrically determined with ABTS as the substrate.

Figure 1: Schematic showing the principle of the Cell Death Detection ELISA^PLUS.

Figure 2: Dose-dependent induction of apoptosis in U937 cells, detected using the Cell Death Detection ELISA^PLUS. U937 cells (10⁴ cells/well, in 200 μl) were incubated with different concentrations of camptothecin (CAM) for 4 hours at 37°C. Before and after lysis, cells were centrifuged and the supernatant was analyzed. Results were plotted as dose vs. response.
Result: Amounts of cytoplasmic oligonucleosomes (an indicator of apoptosis) increase as CAM concentration increases. Cell culture supernatants removed from the cells after treatment (but before lysis) gave no signal, indicating that there were no necrotic cells during the treatment.