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 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.
Our team has developed assays for a number of 7 Transmembrane Receptors (7TMR) (or GPCR) targets, conducting high throughput screens and mechanistic studies.
We have 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).
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.
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.