This tutorial will give an overview of the principles of optical sensor-based label-free technology and how labeled and label-free technologies can be used together, helping researchers to:
- measure the pathway-independent response from cellular interactions that occur during signal transduction, allowing observation of the entire biological picture,
- combine label-free analysis with orthogonal labeled technologies to comprehensively identify and characterize target and ligand behavior,
- gain novel information about ligand pharmacology, unseen using labeled technologies,
- use a single batch of cells for sequential label-free and labelled assays by exploiting the noninvasiveness of label-free, enabling maximum return from precious cells, and
- manage a workflow of lower throughput SPR systems.
Principle of Operation
Cellular Assays. Optical label-free technology, of which Corning Epic® is an example, measures changes in light refraction resulting from dynamic mass redistribution within the cell. This occurs in response to receptor activation or deactivation in a zone within the cell’s monolayer. The response is indicated by a wavelength change (red to green), in the emitted light.
Labeled technologies such as fluorescence, however, measure a particular biomarker within an individual signaling pathway (Figure 1).
Biochemical Assays. Label-free biochemical assays work similarly except that the change in measured mass is due to analyte binding rather than redistribution in cellular assays.
In summary, any cellular activity will be recorded regardless of the signaling pathway activated. In a biochemical assay, the inherent high sensitivity will allow detection of very weak interactions.
Characterizing GPCR Signaling
Our experiments have shown that label-free confirms the functional pharmacological profile (i.e., agonist, antagonist, inverse or partial agonist or allosteric modulator) of a particular ligand receptor interaction as shown by comparable labeled assays, regardless of exact potencies. For well-studied, canonical ligands and receptor targets, a ligand showing agonist behavior in a labeled system will present agonist behavior in label-free.
There is no reason to expect exact correlation between potencies in labeled and label-free analysis. In fact, there will probably be variation due to major differences in how the two platforms record cellular behavior. One of three things can happen when studying a ligand’s behavior using labeled and label-free assays: potency determined by the label-free is higher; potency determined by the labeled assay is higher; they are equivalent.
While ligand potency values may differ from label-free to labeled systems, the rank ordering of potencies between the two should be consistent. This has been shown in many publications.
Each outcome gives important information about both the pathway-dependent and global events upon receptor activation and is an important value-add for researchers. In two studies, a label-free assay pointed to activation of the Gq signaling pathway, which the labeled calcium assay confirmed. In another two studies, label-free pointed to activation of the Gi pathway, which was then confirmed by the labeled AlphaScreen™ cAMP assay (PerkinElmer). Importantly, a single label-free assay will indicate if no activation has occurred, which a labeled assay may not.
In addition to eliciting similar pharmacologies and rank order of potency profiles to labeled assays, label-free technology has generated responses that have gone undetected in a conventional labeled second messenger assay. A recent publication compared the GPCR HTS screening results from 100,000 compounds generated using an industry-standard calcium flux technology (FLIPR) with that of a Corning Epic label-free assay.
Although most compounds tested were inactive, FLIPR-specific and Epic-specific hits were identified. Using label-free as a follow-up, the majority of the FLIPR-specific hits were shown to be false positives. Label-free technology can, therefore, be used as an orthogonal screen to eliminate HTS false positives identified in second messenger assays before progressing into hit profiling. Conversely, luminescent aequorin technology was shown to identify specific hits unseen by optical label-free technology. These results emphasize the need for robust orthogonal label-free and labeled technologies for comprehensive target and ligand characterization.