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Tutorials : Apr 15, 2011 ( )
Combining Labeled and Label-Free Tools
Advantages Include a More Complete Picture of Intricacies of Cellular Systems!--h2>
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:
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.
Maximizing Useful Data
Figure 2 demonstrates the noninvasive nature of label-free technology. The PerkinElmer ATPlite® cytotoxicity assay results, demonstrated that the EnSpire label-free platform had no effect on cell viability. ATPlite was run on endogenously expressed receptors in cells that had undergone a 60-minute label-free scan, both pre- and post-receptor activation, and also run on identical cells that did not undergo label-free analysis. There was no measurable cytotoxicity relative to the positive control showing that cells can be re-used after label-free analysis.
This means that one batch of cells can be used to run two assays. As production of cells is both a laborious and costly step in the research workflow, running label-free assays combined with, for example, luminescence or fluorescence assays, using the exact same batch of cells is a real benefit.
Surface plasmon resonance (SPR) is a label-free technology that has proven powerful in characterizing protein:ligand interactions. Interacting species are immobilized onto a surface and as interaction partners bind to immobilized molecules, refractive index at the surface alters in proportion to the change in mass concentration. Changes in SPR signal over time are then presented graphically.
The results can be used to display the formation and dissociation of complexes over the entire course of an interaction, with the kinetics (association (ka) and dissociation (kd) rates) shown by the shape of the reaction curve. The affinity (binding strength KD) of the interaction is also an available output of this technique.
Optical label-free generates comparable results to SPR for binding strength. This offers an exciting opportunity to use the two techniques in a complementary way. Our experiments have shown how molecular interactions between carbonic anhydrase and a number of low molecular weight molecules were reliably detected with picometer shifts down to around 10 pm.
Furthermore when KD values were calculated and compared to values determined by a market-leading SPR system, the values were in broad agreement across the molecular weight range as shown in the Table.
When nonplate based SPR becomes a throughput bottleneck, the EnSpire with Epic label-free technology can help as it’s a lower cost plate-based system. By using the Epic technology to screen based on binding strength, (KD), the compounds that demonstrate binding can progress into SPR for determination of ka and kd rates. This lower sample number becomes manageable for the SPR system. The two systems are complementary.
In summary, combining optical label-free technology with classical labeled methods gives a more complete picture of cellular systems, while combining different types of optical label-free technology such as Epic technology and SPR together can bring throughput and workflow benefits to biochemical protein:ligand binding, thus helping to drive research forward.
Tim Cloutier, Ph.D., is global applications team leader, Janet Park is global product leader, and Paul Butler ([email protected]) is market development leader at PerkinElmer.
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