Complement-dependent cytotoxicity (CDC) is a mechanism by which antibody-coated target cells recruit and activate components of the complement cascade, leading to the formation of a membrane attack complex (MAC) on the cell surface and subsequent cell lysis.
Creative Biolabs has extensive experience with CDC assays and can help you understand the interaction between your compound or biologic and the complement system, taking you to clinic faster. We conduct CDC bioassays on behalf of clients, either as a standalone project or as part of a larger development project. We are committed to developing the most appropriate experiments to help you understand the molecule.
In our CDC assay, the expressing target cells are incubated with antibodies or ADCs in human serum containing active or heat-inactivated complement. The dead cell protease activity released from cells that have lost membrane integrity is measured as an indicator of cell lysis.
The CDC mechanism works in a similar manner to ADCC. Antibodies bind to target cells through specific antigens and activate multiple ways of attacking target cells, called the complement cascade, rather than recruiting NK cells. The binding of complement leads to the induction of membrane attack complexes, leading to cell lysis.
The current gold standard target marker for antibody-mediated cytotoxicity determination is the stable isotope Chromium-51 (51Cr). Before performing the assay, the isotope is preloaded into the target cell. After lysis, target cells loaded with 51Cr release 51Cr into the supernatant. The radioactivity measured in the supernatant indicates the degree of cell lysis. Radioactive analysis such as this brings obvious safety threats and waste disposal issues. The labeling process can also be arduous and time-consuming. In addition, artifacts and chromate ion toxicity during labeling are also possible.
Creative Biolabs’ recently developed cell-based methods bypass the need for radioisotope-labeled cells and no longer rely on measuring molecules released by lysed target cells, or relying on antibody binding to the target or FcγRIII receptor. Our CytoTox™ bioluminescent ADCC and CDC analysis use the same conventional ADCC and CDC processes that induce target cell apoptosis. This lysis causes the cytoplasm to release endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolysis and gluconeogenesis pathway. By adding a coupling enzyme, adenosine triphosphate (ATP) is generated, and when coupled to a luciferase/luciferin reaction, a measurable bioluminescence signal is generated. This signal is directly proportional to the amount of GAPDH released and the amount of target cell lysis. CytoTox™ analysis is non-destructive, homogeneous, and easy to adapt to automated formats. In addition, because GAPDH is a natural cellular component, there is no need to pre-label, transfect, transform or otherwise introduce the molecule into the target cell.
Fig.1 CytoTox™ assay principle.
Accuracy and precision are critical to the development of bioassays and novel biological agents. Our CDC Reporter bioassay is both accurate and precise, and has been prequalified in accordance with ICH guidelines, and shows the precision, accuracy and linearity required for routine use in potency and stability studies. We work closely with you to choose the most suitable method based on the immune cells or diseases of interest.
Fig.2 Cryopreserved NK Cell Test Data.
Fig.3 CDC assay optimization and validation data.
For more detailed information, please feel free to contact us or directly sent us an inquiry.
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
All products and services are for Research Use Only. Do Not use in humans.
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Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.