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Development of an Image Based Assay to Detect C5a Receptor Occupancy

Imbert, Pierre-Eloi, Siebert, Daniela, Unterreiner, Vincent, Meyerhofer, Marco, Leder, Lukas, Mcallister, Kathryn, Gabriel, Daniela and Parker, Christian (2013) Development of an Image Based Assay to Detect C5a Receptor Occupancy. Abstracts for MipTec 2010.

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Stimulation of the C5a receptor is the final common step in the three different pathways leading to activation of the complement cascade. Activation of the complement pathway has been implicated in a number of different diseases including; rheumatoid arthritis (RA), psoriasis, ischemia/reperfusion (I/R) injury, glomular nephritis and possibly even sepsis. There is strong evidence that activation of the complement cascade, by the alternative complement pathway, is involved in Age-related Macular Degeneration (AMD). Variants in two key regulators of the complement cascade, factor H and factor B, lead to an over activation of the alternative complement pathway, accounting for up to 75% of AMD cases. In addition, leukocytes, mainly monocytes and macrophages, cell types known to express C5aR, are seen by histology in neovascularization, Retinal Pigmented Epithelium degeneration, and breakdown of Bruch’s membrane leading to AMD.
The C5a receptor is a GPCR that once activated leads to changes in intracellular signaling which can be detected by a number of conventional assay formats. However, while these assays can detect intracellular signaling events they give no indication as to the location of the receptor, nor do they allow monitoring of receptor binding only the subsequent signaling events.
This poster describes an image based assay which is capable of monitoring the binding of fluorescently labeled C5a to its receptor. This assay not only allows measurement of binding events by monitoring competition binding of the fluorescently labeled C5a but also localization of the ligand bound receptor. The ability to track the location of the ligand bound receptor allows one to visualize receptor internalization over time. In addition, the ability to direct binding of the labeled ligand makes the requirements for antibody staining and washing steps obsolete resulting in an easily automatable protocol.

Item Type: Article
Keywords: C5a receptor assay, assay development, High content screening
Date Deposited: 13 Oct 2015 13:16
Last Modified: 06 Jul 2016 23:45