Tumor cells are characterized by their rampant proliferation potential. Aragen has expertise with a broad range of proliferation assays (e.g. MTT, Cell Titer Glo, BrdU incorporation, etc.) that can be used on a variety of cell types. These assays are applicable for both large and small molecule test agents and can be applied to variety of suspension and adherent cell lines including but not limited to tumor cell lines and primary PBMC’s.
Apoptosis is a programed cell death used by the body to remove damaged or unwanted cells and occurs during cytotoxic T cell killing and with many cancer chemotherapies. As such, it provides an important mechanism of action for the killing of unwanted tumor cells. Apoptosis is characterized by events such as expression of phosphotidylserine on the cell surface, activation of caspase enzymes and the fragmentation of the genomic DNA, loss of membrane integrity and decrease in mitochondrial function. Aragen Bioscience has effectively reduced to practice a variety of such assay enabling molecules with differing mechanisms can efficiently be tested. These assays are typically run using FACS but can also be executed in a plate reader format.
Target Cell Antibody Binding
Most of the protein-based therapeutics that are used to treat cancer bind to cell surface antigens. Hence, robust assays that enable the sensitive and reproducible detection of these binding events if critical for the assessment of potential cancer therapeutics. At Aragen Bioscience, the binding of this class of test agents (antibodies, peptides, other types of ligands) is generally performed using flow cytometry. This method is very sensitive and has single cell resolution which provides many advantages over alternative techniques. Fluorophore-labeled test agent is bound to the cells and fluorescence measured by FACS.
In Vitro Angiogenesis
Rapidly growing tumors require a constant supply of oxygen and nutrients. To do so, the tumor must promote the formation of new blood vessels, a process known as angiogenesis. In vitro angiogenes is measured using tube formation by primary endothelial cells (typically primary HUVEC) cells. Matrices that either promote angiogenesis (e.g. Matrigel) or are permissive for angiogenesis (e.g. collagen gels) are utilized to assess the effect of test agents on angiogenesis. The propensity to form tubes (i.e. In vitro vessels) in this format is evaluated using microscopy. Images are capture and analyzed for tube length as well as the number of cellular nodes within the tubular network.
Cell biology and physiology is a very complex and dynamically evolving field of study. Feel free to contact us with our custom assay development needs.
Effector Function (e.g. ADCC and CDC assays)
The immune system contains many mechanisms for the identification and attack of unwanted cells (e.g. viral infection, tumors, etc.). Amongst these are Antibody Dependent Cellular Cytotoxicity (ADCC ) and Complement Dependent Cytotoxicity (CDC). In both cases, the cellular killing is mediated by an antibody binding to its target on the cell surface. In the case of ADCC, the killing is mediated by natural killer (NK) cells, while in the case of CDC activity, the killing is mediated by complement proteins. In both cases, Aragen has significant experience with a broad diversity of target cells and mechanisms of killing.
For ADCC assays, Aragen typically utilizes IL-2 activated normal PBMC’s as effector cells and a variety of tumor cells as target cells. However, various sources of PBMC’s (e.g normal vs diseased donors) can also be sourced. Aragen’s preferred readout for this assay is a FACS-based method, although alternative methods (e.g. ATP release) can also be used.
For CDC, these assays are set up very similar to the above mentioned ADCC assays using FACS as a readout. However, instead of using effector cells (e.g. PBMC’s) human complement is used to initiate the killing.
Aragen also has a panel of assays to detect mechanisms of non-antibody dependent mechanisms of cell death, e.g. apoptosis.
Membrane TNFα (“mTNFα”) ADCC and CDC Bioassays
ADCC and CDC are important mechanisms used by antibody drugs to kill targeted tumor cells. The key to Aragen Bioscience’s ADCC assay is the development of our mTNFα cell line which expresses a membrane bound (non-cleaved) form of the protein. This mTNFα molecule can bind antibodies and provides a readout for the induction of ADCC and CDC activity. The CDC assay uses normal human serum as the source of complement. By implementing strict QC standards, Aragen Bioscience can provide efficacy and potency profiles of your therapeutic antibodies.