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| Product Name | Cell Death Detection ELISAPlus Kit (Histone-DNA Complexes, Photometric) |
| Catalog No. | CCAT-HMM-0063 |
| Description | A quantitative sandwich enzyme immunoassay for the specific detection of mono- and oligonucleosomes (histone-associated DNA fragments) released into the cytoplasm during apoptosis, before plasma membrane breakdown occurs. The photometric readout makes this kit accessible to laboratories without flow cytometry or fluorescence microscopy capabilities, while providing quantitative, high-throughput apoptosis detection. |
| Intended Use | Quantitative detection of apoptosis in cell lysates; evaluation of pro- and anti-apoptotic agents; chemotherapy and radiotherapy-induced apoptosis quantification; apoptosis detection in samples unsuitable for flow cytometry; high-throughput screening of apoptosis-modulating compounds. |
| Principle / Technology | Sandwich ELISA using two mouse monoclonal antibodies: anti-histone (biotin-labeled) for capture and anti-DNA-POD (peroxidase-conjugated) for detection; the antibodies bind to histone and DNA components of nucleosomes respectively, specifically detecting apoptosis-associated DNA-histone complexes but not free histone or free DNA |
| Detection Method | Colorimetric ELISA; absorbance at 405 nm with reference at 490 nm; microplate format |
| Sample Type | Cell lysates from mammalian cells (adherent or suspension); cytoplasmic fraction (20,000 × g supernatant); 1 × 10^4 to 1 × 10^5 cell equivalents per well recommended |
| Sensitivity / LOD | Detects apoptosis in as few as 50 cells per well; enrichment factor (apoptotic vs. necrotic signal) >30:1; dynamic range 0.05–15 enrichment factor relative to negative control |
| Specificity | Specific for apoptosis-associated nucleosomes; necrotic cells show much lower signal (enrichment factor <2); no cross-reactivity with free DNA, free histones, or intact chromatin |
| Reaction Conditions / Protocol | Lyse cells in 200 µL lysis buffer (30 minutes at room temperature); centrifuge at 20,000 × g for 10 minutes; transfer 20 µL supernatant to streptavidin-coated microplate; add 80 µL immunoreagent mixture (anti-histone-biotin + anti-DNA-POD); incubate 2 hours at room temperature with shaking at 300 rpm; wash 3 times with incubation buffer; add 100 µL ABTS substrate solution; incubate 10–20 minutes at room temperature with shaking; add 100 µL ABTS stop solution; read absorbance at 405 nm |
| Components / Formulation | Streptavidin-coated 96-well microplate (pre-blocked), anti-histone-biotin antibody (100× concentrate), anti-DNA-POD antibody (100× concentrate), positive control (lyophilized apoptotic cell lysate), lysis buffer (10×), incubation buffer (10×), washing buffer (10×), ABTS substrate tablets, ABTS substrate buffer, ABTS stop solution, plate sealing films |
| Storage Conditions | Store at 2–8 °C; antibodies and positive control store at -20 °C for long-term; ABTS substrate protect from light |
| Shelf Life | 12 months from manufacture date |
| Package Specifications | 96 tests (one 96-well plate), 5 × 96 tests, 10 × 96 tests |
| Product Form | Pre-coated microplate with liquid reagents |
| Key Features | Quantitative ELISA format accessible to labs without flow cytometry; high enrichment factor discriminates apoptosis from necrosis; pre-coated streptavidin plate with optimized blocking; includes positive control lysate; total hands-on time ~3 hours; plate-based format enables high-throughput screening; no radioactive reagents |
| Purity | Monoclonal antibodies affinity-purified; endotoxin <0.1 EU/µg; ABTS substrate purity >99% |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Cold pack 2–8 °C; antibodies may ship at ambient for short transit but should be stored at -20 °C upon receipt for long-term storage |
| Expiration Date / Stability | 12 months under recommended storage; reconstituted positive control stable 2 weeks at 4 °C or 3 months at -20 °C; reconstituted ABTS substrate stable 1 week at 4 °C protected from light |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; ABTS stop solution contains SDS — avoid skin contact; not classified as dangerous goods |
| Compatibility | Compatible with cell lysates from human, mouse, rat, and other mammalian species due to conserved histone sequences; lysis buffer is non-denaturing; avoid using PBS with calcium or magnesium for cell washing prior to lysis; compatible with 96-well plate readers with 405 nm filter |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | Always include blank (lysis buffer only), negative control (untreated cells), and positive control (provided or camptothecin-treated cells). Normalize enrichment factor to cell number or protein content. For weak apoptosis induction, increase cell number to 5 × 10^5 per well in a 6-well culture format. Background absorbance at 405 nm should be <0.2 AU for negative control; higher values may indicate spontaneous apoptosis or cell handling damage. |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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