Global Platform for GPCR Investigation
The IP1 quantification takes only one hour, and requires only an off-the-shelf TR-FRET detector available from several suppliers. Using robotic instrumentation, a single instrument can perform 100,000 assays per day (Figure 3).
A significant advantage of IP-One is its close relationship to Cisbio's existing cAMP assay. The two assays run on the same industry-standard hardware and follow exactly the same methodology and procedure. This offers GPCR investigators a platform to examine both of the principal GPCR pathways, covering in practice about 90% of all GPCRs.
Additionally, Cisbio has recently developed an improved dye chemistry that boosts the performance available from its HTRF methodology. For some years the acceptor molecule of choice for HTRF has been XL665, a chemically stabilized version of the alginate pigment APC.
This acceptor has good quantum yield, high stability in the face of a variety of solvents and buffers, suffers very few interferences, and has well-documented immunochemical properties.
However, XL665 is a large and complex protein of over 100,000 Dalton, almost as large as the Mab to which it is coupled when used in HTRF. This can cause occasional problems, particularly steric hindrance.
The IP-One assay introduces this new proprietary acceptor, a small-molecule organic dye called d2. It has the same photophysical properties as XL665, plus a straightforward immunochemistry and negligible compound interference.
Tested in a kinase assay format against a library of almost 15,000 compounds, the results from d2 showed over 95% correlation with those from XL665. When coupled to Cisbio's IP1-analogue reagent, it enhances both performance and stability of the IP-One assay. Cisbio's cAMP assays are also to be upgraded to use the new acceptor.
In summary, IP-One is a powerful addition to the GPCR researcher's armory. With continuing development of even more specific Mabs and improving signal-noise ratios, it can make a substantial contribution to drug discovery.