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Label-Free Screening

Our scientists have in-depth experience of label-free technology applied to cell-based assays gained over the past 6 years using Corning Epic, PerkinElmer EnSpire and SRU Bioscience Bind instruments.

These systems incorporate an optical biosensor into a micro-titre plate to measure the cellular response to a stimulus. Specifically, the systems are able to detect changes in mass at the sensor surface resultant from alterations in cell shape and/or protein recruitment or migration to/from the membrane.

The technology is generic being independent of signaling pathway, it is non-invasive (no molecular biology required) and generates a kinetic response over seconds, minutes or hours resulting in an information rich readout.

Label-Free Screening

Label-free techniques are highly suitable for measuring responses in native or physiologically relevant systems such as primary human and animal cells without the need for over-expression or amplification through genetic modifications. This is of great benefit in understanding the molecular pharmacology of a receptor in its native environment, whereas a recombinant system does not necessarily have the natural coupling or utilise the correct signaling pathway.

Label-free techniques may also be of benefit where there is no simple/standard biological readout e.g. for de-orphanisation of receptors and identification of pharmacological “tool” compounds.

The Aurelia Bioscience team have developed assays for a number of 7 Transmembrane Receptors (7TMR) (or GPCR) targets, conducting high throughput screens and mechanistic studies. Specifically…….

Screening: Utilised the Corning Epic system to screen a number of 7-TMR targets including agonist screens for two dual coupled receptors (Gi/Gq and a Gs/Gi couples)

Published proof of concept: Comparing the output from label-free and calcium flux readouts for a muscarininc receptor antagonist screen in a peer reviewed journal. (Ref: Journal of Receptors and Signal Transduction, 2009; 29(3–4): 163–172)

Comparison of Label-Free versus FLIPR data generated on a Muscarinic Receptor Identifying Antagonist Compounds
100,000 compounds were screened in antagonist mode using a CHO cells expressing a muscarinic receptor.

Inhibition data generated from the label-free assay was compared to data for the same compounds tested in a calcium flux screen using FLIPR.

The correlation of the two sets of data highlighted a group of “hit” compounds that were unique to the label-free assay (Set C)

Label-Free versus FLIPR

Mechanistic studies: We have developed and validated assays for targets in common cell backgrounds and primary human and animal cells where available. Label-free assays have been used as an orthogonal screening approach to more standard screening assays.

Identification of Compounds that Behave as Agonists, Antagonists or Inverse Agonists in a Single Assay.
A recombinant cell line was challenged with compound and the label-free response measured over a period of 15 minutes to determine agonism.

The cells were then stimulated with a standard agonist at an EC80 concentration and any antagonist effect measured over the next 45 minutes.

Circles highlight data points used for XC50 calculation.

Label-Free Agonists Antagonists

Phenotypic screening: Our experience extends to developing assays with human neutrophils to measure responses to a range of stimuli e.g. chemokines. (ref ELRIG 2007 presentation)

Real Time Response in Human Neutrophils to Interleukin-8 (IL-8)
Neutrophils were isolated from human donor whole blood using density gradient centrifugation and plated into 384 well biosensor plate.

IL-8 was added to wells at different doses (shown) and responses measured over a 25 minute period.

Label-Free Neutrophils

Instrument evaluation: Conducted an evaluation of the PerkinElmer EnSpire multi-modal reader. This benchtop instrument is a label-free platform suited for assay development and low to medium throughput screening

EnSpire Multimodal Reader from PerkinElmer
The instrument is able to measure label-free responses in addition to fluorescent and luminescent responses sequentially from the same wells of a plate.

It can therefore be used to multiplex complex cell-based assays measuring kinetic responses over seconds, minutes or hours.

EnSpire Multimodal Reader

Applications for targets other than 7-TMR biology include:

  • Receptor tyrosine kinases e.g. EGFR
  • Ion channels e.g. Potasssium channels
  • Viral proliferation in cells – cell death
  • Cell adhesion – or prevention of
  • Cell proliferation – apoptosis

If you want to understand more about your cellular target by applying a label-free approach then...

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