Biology Reference
In-Depth Information
characterize this, the implementation of multiplexing assay represents a
promising improvement. First of all, it would allow the detection of at least
two events at the same time in the same cell, which would increase the re-
liability between the two tests. In addition, it would also decrease by twofold
the amount of compound used and the time spend for screening the assays
that were combined. HTRF has the peculiarity to be perfectly compatible
with multiplexing. As already mentioned, the lanthanide emission spectra,
and more particularly terbium-based FRET donors, show several peaks that
are perfectly compatible with the excitation of several acceptors. In agree-
ment, one could easily imagine combining both IP-One and cAMP mea-
surements in the same well if, for example, the fluorescent IP is labeled
with a red acceptor and the cAMP with a green one. Of note, TR-FRET
multiplexing has already been reported to monitor the binding of an agonist,
an antagonist, and a coactivator on the estrogen receptor.
82
The receptor is
labeled with terbium, and the agonist and antagonist with fluorescein and
semi-naphthalene fluorescein, respectively. The decrease of the FRET sig-
nal between the receptor and the antagonist could be recorded at the same
time as the appearance of a FRET signal between the receptor and the ag-
onist. Addition of a coactivator labeled with Cy5 gave rise to a concomitant
third FRET signal that was also simultaneously recorded. Alternatively, an-
other report of TR-FRET-based assay multiplexing has been reported by
Horton and Vogel.
37
In this study, they combined two kinase assays, one
using the terbium GFP FRET couple and the other one the europium
AlexaFluor647 couple. As there was good separation between the different
lanthanide emission and acceptor excitation spectra, the authors could
show that both assays could be used together. Such technological solutions
are perfectly conceivable in the field of HTS targeting GPCRs or other
cell-surface receptors.
5.4. Toward further miniaturization
A further improvement of TR-FRET-based assays would be further min-
iaturization. While most of the reported assays were already proved to be
compatible with a 384- and even a 1536-well plate format, a further im-
provement would be to scale down to the microarray format. A first step
in that direction was made when detecting the interaction between
lanthanide-labeled biotinylated BSA immobilized on a chip and Cy5-
labeled streptavidin.
83
Another proof of concept for the use of receptor in
microarrays has been provided by the analysis of the coactivator binding
Search WWH ::
Custom Search