Principles of the Ziva®-Tox Assay

Ziva®-Tox is an ELISA-based ultrasensitive cell toxicity assay that uses a chemiluminescent substrate to detect the presence of BrdU incorporated in actively proliferating cells. The Ziva®-Tox assay determines a Percent Cytotoxic Effect ("PCE") (see Calculation section below) for the treated target cells, by determining the extent of cellular BrdU DNA synthesis inhibition caused by the treatment of the target cells with the cytotoxic agent. This is performed by measuring the extent of BrdU label incorporated into cytotoxic agent treated target cells during or after cytotoxic agent treatment, relative to the extent of BrdU DNA synthesis in non-treated control target cells. BrdU DNA synthesis is very sensitive to perturbations to a large number of healthy cell functions, revealing if cell DNA synthesis is inhibited or impaired in cells undergoing damage or death from a cytotoxic agent. Therefore, the magnitude of the difference in the amount of BrdU cell DNA detected in cytotoxic agent treated cells relative to the amount of BrdU cell DNA detected in untreated control cells is a measure of the magnitude of the cytotoxic effect on the treated cells. By comparing the signal of treated cells with untreated cells, one can derive a measure of the percent of cell cytotoxicity.

Advantages of the Ziva®-Tox Assay

1. Significantly increased dynamic range and sensitivity

   The Ziva®-Tox assay does not have the problem of high spontaneous release of non-specific signal because it directly detects BrdU incorporation into the DNA of surviving cells. Precise biological effects can be measured by comparing the signal of treated target cells to the control wells containing untreated target cells. This results in a much higher dynamic range for the Ziva®-Tox assay and causes the assay to be significantly more sensitive in detecting the appearance of agent-induced cytotoxic effects associated with the treated target cells.

2. More flexibility in the quality of the target cells

   The quality of the target cells have much less of an effect on the assay results and the cell quality can vary greatly because there is no “spontaneous release” or its equivalent associated with in the treated, and non-treated control, target cells. In addition the serum concentration has little effect on the ability to detect cytotoxic effects associated with the treated target cells.

3. Great flexibility in time of cytotoxic agent exposure to target cells

   Cytotoxic agent plus target cell sample exposure times using the Ziva®-Tox assay method are very flexible and the cells can be exposed to the cytotoxic agent for 2-4 hours, days, or weeks, if necessary. This flexibility allows the operator to more fully interrogate the biological response of the target cell to cell-mediated toxicity or potentially cytotoxic agents. Longer incubations, in effect, serve to amplify the interaction between target cells and the cytotoxic cells or reagents. In addition longer exposure times may more closely mimic natural conditions in biological systems.

4. Great flexibility in timing and length of BrdU labeling of target cells

   The Ziva®-Tox assay BrdU labeling method is also very flexible. The labeling of the control and target cells can be performed simultaneously with the cytotoxic agent exposure, or in the final 2-24 hours of the exposure to the cytotoxic agent. Precise biological effects can be measured by comparing the Ziva®-Tox assay signals of treated target cells to the control wells containing untreated target cells.

5. Greater sample size flexibility

   With Ziva®-Tox it is possible to interrogate very small numbers of cells (< 1,000 cells per well). Biological effects have been detected using between 500 to 40,000 cells per sample and a Ziva®-Tox cytotoxicity assay can be done using as few as 50-100 cells or less.

6. Easy and rapid assay protocol

   After the cytotoxic agent treatment and BrdU labeling period, which is dependent on the experimental system, the actual Ziva®-Tox assay procedure time can be performed in less than 1 hour.