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Understanding Assay Performance Metrics
When embarking on the search for a reliable cell-based assay, it's common practice to start by typing "Receptor X cell-based assay" into the search bar of your preferred internet browser. From there, you navigate through the multitude of search results, sifting through the information provided. This process can often feel overwhelming, especially when attempting to discern which metrics and parameters are crucial for comparing and ultimately selecting the most suitable assay for your specific research needs. That's where this blog comes in – to serve as a helpful guide in navigating the complexities of selecting a commercially available cell-based receptor assay from the plethora of available options. From understanding the sensitivity and specificity of the assay to assessing its reproducibility and robustness, each metric plays a vital role in determining which assay is right for your project.
EC50 and IC50 Values
The values of EC50 and IC50 are used to describe the concentration of a drug that produces an activation response (EC50), or an inhibition response (IC50), that is halfway between its baseline and maximal effect.1 In other words, these values represent the concentration at which a drug is exerting 50% of its maximum functional response. EC50 / IC50 values are calculated from dose-response analyses performed using in vitro assays, and they are used in the context of early-stage drug discovery to rank the potency of drug candidates against a specific receptor. As EC50 / IC50 values march lower during the drug optimization process, the more potent a given compound is against the target receptor. It’s important to understand that EC50/IC50 values are not constants. Indeed, they can vary greatly between different assay platform technologies. Therefore, when assessing the relative performances of different commercially available receptor assays, the advertised EC50 (IC50) value for a given validated reference compound is a critically important comparator metric.
Signal-to-Background (a.k.a. Fold-Activation, or Fold-Reduction)
Calculating Signal-to-Background (S/B) is an important method of normalizing raw data that should be calculated in most experimental assays. The term ‘S/B’ is generic, being synonymous with the more specific terms of Fold-Activation (F/A) in agonist-mode assays and Fold-Reduction (F/R) in antagonist-mode assays. In cell-based reporter assays S/B is the ratio of the measured receptor-specific signal from the test compound treated assay wells divided by the receptor-specific background signal from the untreated assay wells.2 In the case of performing an agonist-mode screen of a specific receptor target, using a cell-based luciferase reporter assay in which Relative Light Units (RLU) are the unit of measure, the calculation of S/B is:
S/B (or F/A) = RLU Test Cmpd treated cells / RLU Untreated cells
A high F/A ratio in an agonist assay indicates a strong functional response by the receptor, providing a test compound-induced signal that is much higher than the basal level of receptor-specific signal in the untreated reporter cells. High F/A ratios are always desirable and are one hallmark of a robust agonist assay.
Z’
Z' (pronounced "Z prime") is a second hallmark of assay robustness. It is a statistical score commonly used to assess the suitability of an assay for either low- or high-throughput screening applications. What makes the Z’ score so powerful as a diagnostic measure is that it incorporates the two critical assay variables: Standard Deviation (SD) and Signal-to-Background (S/B).2
Z’ = 1 - [3x (SD Test Cmpd treated cells + SD Untreated cells) / (SB Test Cmpd treated cells – S/B Untreated cells)]
The resulting score is a unitless measure that will range between the theoretical values of ‘0’ (or -∞) and ‘1’. The higher the Z’ score, the better the performance quality of the assay. An assay that yields a Z’ between 0.5 and ‘1’ is good-to-excellent and is of sufficient quality for HTS applications. (It bears keeping in mind that a score of ‘1’ is, in practice, unattainable and therefore is a theoretical maximum. It would mean that the assay has no SD in either the experimental or background assay wells.) In contrast, an assay that yields a Z’ value below 0.5 is of poor quality, suffering from high SD or low S/B, or both. Such assay performance is unsuitable for screening purposes at any scale.
Z’ score or 0.5 → ‘1.0’ = good → excellent assay quality; suitable for drug screening
Z’ score between <0.5 → ‘0’ = poor → failed assay; unusable as a screening tool
In summary, Z' is an important statistical measure of assay robustness. It provides researchers with the means to make a quantitative assessment of assay quality when comparing different commercial offerings or platform technologies.
INDIGO Biosciences provides cell-based receptor reporter assays that are highly optimized. We go to extraordinary lengths to offer assays that deliver best-in-class performance metrics, boasting high S/B and Z’ scores, and low EC50/IC50 metrics. INDIGO offers researchers both all-inclusive assay kits as well as assay services. Next time you embark on the search for a reliable cell-based assay, give us a look.
References
- Singh, A., Raju, R., Mrad, M., Reddell, P., & Münch, G. (2020). The reciprocal EC50 value as a convenient measure of the potency of a compound in bioactivity-guided purification of natural products. Fitoterapia, 143. https://doi.org/10.1016/j.fitote.2020.104598
- Zhang, J.-H., Chung, T. D. Y., & Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. SLAS Discovery, 4(2), 67–73. https://doi.org/10.1177/108705719900400206
