Application note: Characterising chemokine receptor inhibitors with Alpha SureFire Ultra assays

In this application note, PerkinElmer discuss how their ValiScreen CXCR2 GPCR cell line (#ES-145-C) was utilised to examine the downstream effects of chemokine receptor modulation using multiple blocking antibodies and a small molecule…

The ideal research platform for inhibitor development is able to screen for both large and small molecule inhibitors within the same assay format. Assays developed for the detection of small molecule inhibitors may not be amenable to detection of blocking antibodies. One way to overcome this is to utilise assays that probe the downstream signalling events that are impacted by both types of inhibitors. As such, it is important to have available simple, fast and sensitive assays for the measurement of antibody modulation of receptors in a cellular system.

The measurement of protein phosphorylation is a useful tool for measuring the modulation of receptor activation by both antibodies and small molecules. Alpha SureFire Ultra assays are mix-and-read assays that use the highly sensitive Alpha bead-based proximity detection system, coupled with CaptSure immobilisation technology, to assess relative levels of both phosphorylated and total states for a variety of proteins. These assays do not suffer from interference from exogenous antibodies. As there are no wash steps, these assays are automation friendly, may be applied to both small- and large-scale screens, and used to assess phosphorylation status in complex matrices including diverse cell culture models and serum samples.

To demonstrate the utility of Alpha SureFire Ultra assays for measuring a variety of inhibitors to different cell surface receptors, we chose to examine CXCR2 pathways in a cellular model system. Assay conditions were optimised to measure the blockage of this receptor (activated with appropriate agonists). Receptor activity modulation was assayed by the detection of ERK and AKT phosphorylation status using the Alpha SureFire Ultra assays.