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). Continuing to the present day, the CRA is considered to be unparalleled in sensitivity.
Flow-based detection of degranulation markers, or ELISA-type indirect measurements of NK killing, have become common more recently, but ImmunoSpot's Target-cell Visualization Assay has shown equivalent sensitivity and reproducibility to CRA, while providing high-throughput screening capabilities no other method can match.
The 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 fluorescently-labeled target cells simultaneously, and can be performed in miniaturized plate format(s), thereby reducing the number of effector cells needed by up to 30-fold.
The TVA™ Platform utilizes fluorescent image cytometry, measuring the intensity of stained target cells, and the resulting reduction in this intensity as targets are lysed, to quantify NK-mediated lysis of target cells.
TVA is far less laborious than CRA, has shown high reproducibility, and has been validated in clinical testing labs, with fully-automated analysis and data reporting and export. Streamlined quality control and tamper-proof audit trails facilitate the workflow in regulated clinical laboratories and cell production facilities.
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.
TVA™ vs. Chromium Release Assay (CRA)
PBMC were used as effectors with labeled K562 as target cells. Both assays were equally sensitive in a 96-well plate assay, however, the TVA™ Platform is far less labor-intensive and requires a fraction of the investigator’s time. The TVA™ has no background noise, high inter-assay reproducibility, and provides audit trails and data security for GxP compliance.