Abstract
A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.
Highlights
Antibodies are able to recognize and destroy targeted cells, such as those corresponding to tumors or viral infections, through complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) [1,2]
Cell lysis by the CDC or ADCC process is typically measured for a bulk population by monitoring the release of chromium-51 (51Cr) [3] that had been previously taken up by the cells, or by the release of lactic acid dehydrogenase (LDH) [4,5]
This method can lead to large sample errors in ADCC assays since both the target and effector cells contain LDH [8,9]
Summary
Antibodies are able to recognize and destroy targeted cells, such as those corresponding to tumors or viral infections, through complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) [1,2]. This method can lead to large sample errors in ADCC assays since both the target and effector cells contain LDH [8,9]. All of the available methods provide an average toxicity value for a whole population of cells, providing no information about individual cell behavior These techniques require the evaluation of relatively large cell populations to obtain usable reproducibility. This can be especially problematic in cases where targeted cells are in short supply, such as the use of blood samples from specific leukemia patients
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