Cellular Immunology Assays
Immunophenotyping & Lymphocyte Subset Characterization
Our scientists have expertise in performing assays in whole blood, PBMCs and purified immune cell subsets. Multicolor (up to 5 color) immunphenotyping and functional analysis of immune cells is an area of particularly strong experience at Aragen. In addition to standard flow cytometry, Aragen has the capability of high-throughput 4-color flow cytometry (with the IntelliCyt iQue system) for many single and multiplexed assays. As an example of a multi-panel, multicolor immunophenotyping project, the panels of antibodies shown below were used to stain whole blood samples to determine the effect of test antibodies on the frequency and absolute number of specific cell subsets in peripheral blood.
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.
Immune cell activation is critical for an effective immune response to disease or infection. Aragen routinely performs assays to measure immune cell activation using multiple readouts, alone or in combination. Cells can be activated by general means (e.g. PHA, ConA) or methods activating specific cell subsets (e.g. CD3/CD28 or anti-IGM). Measurements of activation can utilize activation marker expression (e.g. CD69, CD25, CD71, CD154), protein phosphorylation, intracellular or multiplexed cytokine secretion, lymphoproliferation (e.g. BrdU incorporation, CFSE tracing, Ki-67 or PCNA expression) and others. These assays are performed in human whole blood, PBMCs or purified subsets and mouse splenocytes or whole blood. Whole blood provide a particularly useful format for pharmacodynamic assays.
Mixed Lymphocyte Reaction (MLR)
Mixed lymphocyte reactions (MLR) are a common method to assess the cellular immune response. When lymphocyte populations from two individuals, (e.g. two humans or two different mouse strains; “two way” MLR) are co-cultured, T cells that recognize the foreign cells from the other strain become activated and proliferate. “One-way” MLR assays can be performed by pretreating one population of cells with mytomycin C, which restricts their proliferation and allows them to serve as stimulator cells. Aragen Bioscience, has experience with the evaluation of various types of compound is various MLR assay configurations.
Cytotoxic T Lymphocyte (CTL) & Natural Killer Activity Assays
The protection from viral infections and cancers often occurs through Cytotoxic T lymphocyte (CTL) responses. CTL assays are commonly used to quantify cytotoxicity, particularly in the study of tumor and viral cytolysis. The biology around these reactions is very complex and the handling of the immune cells must be done with great care. Otherwise the results (e.g. CD8 cell activation, cellular lysis) can be confusing. Hence, Aragen’s experience is critical to the delivery of robust data for such assays.
ELISA assays are sensitive, precise and quantitative assays that can be used to measure an antigen or an antibody, or generally, any macromolecule that binds another molecule or cell. Aragen Bioscience utilizes colorimetric, fluorescent, time-resolved, and homogenous (requiring no washing step) ELISA formats.
Enzyme-linked immunospot (ELISpot) assays are highly sensitive immunoassays that measure the frequency of individual cytokine-secreting cells. In these assays, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Proteins, such as cytokines, that are secreted by the cells will be captured by the specific antibodies on the surface. After an appropriate incubation time, cells are removed and the secreted molecule is detected using a detection antibody in a similar procedure to that employed by the ELISA. The detection antibody is either biotinylated and followed by a streptavidin-enzyme conjugate or the antibody is directly conjugated to an enzyme. By using a substrate with a precipitating rather than a soluble product, the end result is visible spots on the surface. Each spot corresponds to an individual cytokine-secreting cell.
Characterization of Fcγ Receptor Binding
The team at Aragen Bioscience has developed assays to detect binding of innovator and biosimilar therapeutic antibodies to a broad range of Fcγ receptors and specific variants using ForteBio’s biolayer inferometry (BLI) technology to measure on and off rates.
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.