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The STEP technology has
been applied to many different type of assays.
Calcium
Mobilization Assay on a FLIPR-384 System
GPCR Assay Using Transcription Reporters
Multiplexed Assay
Transporter Assay
siRNA Assay
Calcium Mobilization Assay in FlexStation or FLIPR-384 System
STEP can be directly used in
high throughput drug screening targeting G-protein coupled receptors (GPCRs). Various assays for GPCR
have been established. With the high co-transfection efficiency of STEP
technology, the expression levels of receptors and G-proteins can be
fine tuned in order to produce robust and reproducible assays with
physiological significance. Below is an example of GPCR Calcium
mobilization assay for Orexin 1 receptor in Hek293T cells using STEP.
HTS Statistics for a GPCR
Screening with FLIPR using STEP plates:
| No. of plates |
10
|
| No. of compounds |
3520
|
| % Activity (mean) + SD |
103.0 + 20.1
|
| Threshold for positives (% activity) |
42.7
|
| Number of positives |
112
|
| Number of positives (% of compounds) |
2.6
|
| SD of high controls (% activity) |
6.4
|
| SD of low controls (% activity) |
3.7
|
| Z'-factor (all controls) |
0.70
|
| Z'-factor (mean of intraplate Z'-factor) |
0.70 +
0.05
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GPCR Assay Using Transcription Reporters
In addtion to the calcium
mobilization assay, transcription reporters can also be used to detect
GPCR activity in STEP-based assays. GPCR assays using reporter genes in
various cell lines have been developed with STEP. In the figure below,
an Orexin 1 receptor assay in neuronal SH-SY5Y cells using STEP is
shown.
Orphan Receptors Mediated
Activation of Transcription Factors
Multiplexed Assay
With the high efficiency of STEP
technology, multiple targets and/or read-out can be combined in the
same well without sacrificing sensitivity. Below is an example of
multiplexing two assays for two targets (OX1R and MC4R). The activities
of each receptor can be detected distinctively using Calcium
mobilization or SEAP reporter gene assays.
Alternatively, different receptors have be successfully multiplexed in assays with the same read-out to increase HTS efficiency.
Transporter
Assay
Various transporter assays
have been established using STEP and a couple examples are shown below.
The assay for human peptide transporter, hPepT1, using STEP shows a
highly reproducible and robust biological response. Similar assay for
hPepT2 has also been developed using STEP technology.
Cytotoxicity Assay of a Drug in the Presence
and Absence of a Transporter:
SiRNA Assay
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