The ssDNA Apoptosis ELISA Kit is a convenient, sensitive method for early detection of apoptosis.
Principle:This procedure is based on the selective denaturation of DNA in apoptotic cells by formamide, and detection of denatured DNA with monoclonal antibody to single-stranded DNA (ssDNA). Formamide is a gentle agent that denatures DNA in apoptotic cells, but not in necrotic cells or in the cells with DNA breaks in the absence of apoptosis1. The sensitivity of DNA in apoptotic cells to formamide is not related to DNA breaks, but rather reflects changes in chromatin associated with apoptosis, such as chromatin condensation and digestion of proteins stabilizing DNA. The assay includes attachment of cells to 96-well plates, treatment of attached cells with formamide, and staining of ssDNA in apoptotic cells with a mixture of primary antibody and peroxidaselabeled secondary antibody. The protocol based on the one-step detection of ssDNA with antibody mixture has higher sensitivity and lower number of steps than standard two-step immunostaining2. This mixture is included in the kit in a ready to use form.
The assay can be performed in two formats:1. Cells are grown, treated with apoptosis inducing agents, stained and analyzed in the same microtiter plate. This assay is suitable for high throughput screening when induction of apoptosis is used as the endpoint of drug activity. 2. Cell suspensions obtained from cultures or tissues are transferred into a microtiter plate for staining and analysis. For this assay, cells could be fixed with methanol and stored before transfer into plates. Alternatively, fixation could be performed in microtiter plates after transfer of non-fixed cells. Total assay time is 3–4 hours, which includes fixation and staining. Only one washing step is required. The ssDNA Apoptosis ELISA is for research use only. Not for use in diagnostic or therapeutic procedures.
Assay Characteristics Sensitivity:The high sensitivity of the ssDNA Apoptosis ELISA is characterized by1. Detection of 500 apoptotic cells per well and a linear relation between the absorbance and the number of apoptotic cells in the range of 500–5000 cells. 2. The ability to produce intense apoptosis signal by drug concentration inducing 50% growth inhibition, indicating that the assay sensitivity is sufficient for drug screening2. Specificity for Apoptosis over Necrosis The assay is highly specific for apoptotic cells as demonstrated by the absence of mAb reactivity with necrotic cells or with cells containing DNA breaks in the absence of apoptosis. Specificity of the assay was demonstrated using necrosis induced by hyperthermia or detergent and DNA breaks induced by hydrogen peroxide. These cells were completely negative by formamide-mAb assay, but positive by TUNEL1,2. Specificity is a major advantage of this anti-ssDNA mAb assay in comparison with the widely used TUNEL procedure. Universal Detection The detection of apoptotic cells with formamide-mAb procedure is based on the staining of condensed chromatin in apoptotic nuclei. Since chromatin condensation in compact masses is the most specific and definite hallmark of apoptosis, this method provides universal detection of apoptosis. The apoptotic events that occur without DNA breaks or without activation of specific caspases will be detected with our mAb to ssDNA.
Kit Components:D3878-25A. Antibody Mixture, 1x10ml. Ready to use. Primary monoclonal to ssDNA and HRP-labeled Anti-mouse IgMD3878-25B. Formamide, 1x5ml. Ready to use
D3878-25C. Positive Control, 1x2ml vial, 0.3ug/ml. Single-stranded DNA
D3878-25D. Wash Buffer Concentrate (10X), 1x10mlD3878-25E. ABTS,1x12ml. Ready to use solution of 2,2'-AZINO-bis (3-ethylbenziazoline-6-sulfonic acid) in a proprietary buffer with enhancer. D3878-25F. Stop Solution, 1x12ml HCl solution.
Storage and Stability:Once the kit is opened, Antibody Mixture and ssDNA Positive Control should be thawed on ice, aliquoted and stored at-20º C. Formamide, Wash Buffer, ABTS Solution and Stop Solution can be thawed and stored at 4°C.
