For decades, the Chromium Release Assay (CRA) has been considered the gold standard for measuring Natural Killer (NK) cell-mediated lysis of tumor cells, or of antibody-coated target cells in antibody-dependent, cell-mediated cytotoxicity (ADCC). Until now, the CRA has been deemed to be unparalleled in sensitivity. No longer!
The CTL non-radioactive Target cell Visualization Assay (TVA™) not only has the same sensitivity as CRA, but can also measure the lysis of up to three different target cells simultaneously and be performed in Terasaki plate format, thereby reducing the number of effector cells needed by 30-fold. The TVA™ Platform is based on direct, single-cell imaging, is less laborious than CRA, and has fully-automated analysis and data reporting. Streamlined quality control and tamper-proof audit trails facilitate the workflow in regulated clinical laboratories.NK-Activity ePBMC®NK-TVA™ vs. CRA NK-TVA™ PowerPoint Presentation NK-TVA™ Poster
Assay Principle and Sensitivity
The TVA™ utilzes direct imaging of fluorescence-labeled target cells instead of radioactive chromium labeling Labeled tumor cells are co-incubated at various ratios with Peripheral Blood Mononuclear Cell (PBMC) populations, or other NK cell-containing isolates. Following NK-mediated lysis, target cells lose their fluorescent signal. The direct visualization of remaining viable target cells at the end of the assay determines the percentage of cytotoxicity for each effector to target (E:T) ratio. Up to three differently labeled target cells can be tested simultaneously.
When performed in paralled, the TVA™ and CRA exhibited similar killing percentages irrespective of whether cryopreserved PBMC or freshly-isolated PBMC were used as effector cells, with labeled K652 tumor cells as target cells. Both assays were equally sensitive in a 96-well plate assay, however, the TVA™ Platform is far less labor-intensive, requires a fraction of the investigator's time, and eliminates the need for radioactive chromium. The TVA™ has no background noise, has high inter-assay reproducibility, and provides audit trails.
A major drawback of traditional cytotoxicity assays is that they require large numbers of effector cells to detect cytotoxic effects. The TVA™ can be performed in a miniaturized format, requiring only 1x105 PBMC for assessment of seven E:T ratios in Terasaki plates run in triplicate. The measured percentage of lytic activity is similar to that observed with 96-well plate formats.