The Z prime value (Z´)

Scientists depend on high-quality biological assays for reliable and fast measurements. But how do you ensure the quality of these assays, especially if you are working at scale? Z prime value or Z prime factor, which is also referred to as Z’, is one crucial parameter for measurements in the life sciences particularly for data acquisition in high throughput screening in drug discovery. But what is the Z prime value exactly and how is it determined? In addition, what is its relation to other parameters for assay quality including Z value, Z factor and its other counterparts?

In this blog, we focus on the definition and uses of the Z prime value. We also look at some practical applications amenable to microplate readers, including high throughput screening, and consider some recommendations for the use of Z prime statistics.

Biological assays find many uses in the life sciences including applications in drug discovery, biotechnology and molecular biology as well as analytical areas like bioanalysis and quality control. When they want to focus on the quality of an assay or method for these types of applications, scientists use different statistical parameters. Fine tuning assays based on quality metrics can be the difference between coming up with a crucial scientific discovery or the availability of a life-saving drug for a patient. 

What is the Z prime value (Z’)?

In the life sciences, few people have written exclusively about Z statistics including Z prime or the Z prime value in the peer-reviewed scientific literature. One of the most cited papers however remains a publication by Zhang and coauthors from 1999.¹ This scarcity probably reflects the overall acceptance of the utility of Z statistics and their widespread use in laboratories across the world, particularly in the context of assay development and screening in drug discovery.

The main Z statistics used in the evaluation and validation of high throughput screening and other assays are Z prime value and Z value. In the literature, these are often referred to as Z’ and Z, respectively. In other cases, they are referred to as Z prime factor and Z factor.¹ In this blog, we will refer to these parameters as Z prime value and Z value for simplicity. 

The key differences between the Z prime value and Z value are outlined in Table 1. Essentially, Z value statistics provide a useful tool for comparison and evaluation of the quality of assays during or after high throughput screening and they include test samples. In contrast, Z prime value statistics evaluate the quality of an assay before testing samples. Z prime is based on positive and negative controls only and no test samples are involved.

Table 1. Key differences of Z value and Z prime value.

The differences between Z prime value and Z value become clearer by considering the equations for each of these parameters. 

How do you calculate Z prime value and Z value?

Z prime value is defined by the following equation: 

where µ is the mean and Ϭ is the standard deviation of the signals for the positive and negative controls. As outlined in Table 1, the Z prime value allows assessment of the quality of an assay based solely on controls without considering test samples.

In contrast, Z value evaluates the quality of an assay during or after the testing of samples and is defined by the following equation:

where µs and µc are the means of the signals for the sample and control, respectively, and Ϭs and Ϭc are the standard deviations of the signals of the sample and control, respectively.

Z considers the dynamic range of the assay signal, data variation due to the sample measurement, and data variation due to the reference control measurement. The Z statistic is therefore used during or after high throughput screening to evaluate the actual performance of the assay with test compounds.

Best practice: Z prime usage

In practice, an assay should first be optimized according to the Z prime value (Z’) for conditions such as reagents, procedure, kinetics, instrument, and factors other than those related to the test compounds. This ensures the assay format will have sufficient dynamic range and signal variability and that it will provide useful data. If the Z prime value is small (negative or close to zero), it usually indicates that the assay conditions have not been optimized or that the assay format is not feasible for generating useful data.¹

After this step, the properties of the compound library used for a high throughput screen should be considered and Z value determined. Z values between 0.5 and 1.0 indicate excellent performance. Values between 0 and 0.5 may be acceptable. Values less than zero indicate the assay is not useable.

While the Z prime value is calculated for control data before screening and the Z value is determined for data during screening, the recommended numerical criteria in both cases are mostly the same (in practice, the control in a good screening library may contain a small number of active compounds. Typically, this number is too small to significantly affect the Z prime value. However, if the number of active compounds in the control is too high the Z value and Z prime values may diverge).

In all cases, the Z value is less than or equal to Z prime value for all large datasets if the positive and negative reference controls are properly selected which means that Z prime value can be used for quality assessment in assay development and optimization.¹

What assays benefit from Z prime value?

Examples of assays that benefit from Z prime value evaluation include enzyme activity assays (e.g. kinase inhibition assays), gene reporter assays (luciferase, green fluorescent protein), cell viability assays (CellTiter-Glo, MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, resazurin) and binding assays (for example those that require detection technologies like fluorescence resonance energy transfer or FRET and time-resolved FRET or TR-FRET). Researchers may want to look at protein-protein interactions or protein-ligand binding including to take one example studies of G-protein coupled receptors. They may also be interested in toxicity assays or functional assays (ion channel activity, cytokine secretion or immune cell activation). Many of these assays can be performed at high throughput suitable for drug discovery and development.

While Z prime values are most often used for high throughput assays, they may also be applied to assays that do not require such scale and speed. They find uses in assay validation, optimization and standardization in smaller scale experiments that may involve, for example, different types of enzyme activity measurements. New assay development, ensuring an assay delivers precise measurements, or comparing assay conditions to improve performance parameters like signal-to-noise ratios all benefit from Z prime value analysis.

Some clarifications

The Z prime value does not always need to be greater than 0.5.

It is important to note that while the criterion that Z prime value should be greater than 0.5 has played a significant role in ensuring the quality of many high throughput assays, it may not always be the appropriate cut off for all assays.² Some authors have noted that the widespread use of this cut-off may pose an unwanted barrier for some essential assays to advance to high throughput screening. Cell-based assays are a good example.³ Since cell-based assays are more variable than biochemical assays it seems unreasonable to insist on a Z prime value greater than 0.5.

It is therefore prudent for scientists to take a more nuanced approach to using Z prime value to assess assay quality. In particular, the selection of a threshold for Z prime value should be made in the context of the unmet need for the assay. Decisions should therefore be made on a case-by-case basis.

Other parameters for quality

Z statistics are certainly not the only criteria for assay quality in high throughput or other settings. Other assay parameters like signal-to-blank, signal-to-noise, dynamic range and limit of detection also play essential roles in ensuring the quality and performance of biological assays. Each should be considered depending on the context and need. You can read more about other parameters for determining assay quality in the BMG LABTECH eBook Microplates in action: Recommendations for use. 

Z prime values on microplate readers

The choice of a high-quality microplate reader is vital for achieving excellent Z prime values and reliable assay results. Microplate readers offer opportunities to perform biological assays quickly, accurately and at high throughput. They provide high sensitivity, low noise, and consistent performance across wells, which are critical for accurate and reliable Z value statistics. Microplate readers specifically developed for high-throughput screening purposes provide top-of-the-line sensitivity and detection speed with reduced variability. They can handle a wide range of microplate and assay formats and are designed to seamlessly integrate into robotic automation systems. These features collectively enhance the efficiency and quality of screening processes and of data generation.

Some microplate-based applications of Z prime values

In the application note GPCR activation is measured using Cisbio's cAMP and IP1 HTRF HTplex cell-based assay Lumi4-Tb™ HTRF^® (homogeneous time-resolved fluorescence technology) was used to measure both cAMP via green readout (520 nm) and inositol phosphate 1 (IP1) via a red readout (665 nm). Assays were performed with a microplate reader suitable for high throughput screening using simultaneous dual emission detection. The quality of both assays for these high throughput analyses was confirmed by determining the Z prime value (Fig. 1). Both assays gave Z prime values greater than 0.75 which confirmed their suitability for use in a high throughput environment. The assays were used to study the activation of the G-protein coupled vasopressin-2 receptor in Chinese Hamster Ovary (CHO) cells.

The application note Increasing throughput with dual emission AlphaLISA-AlphaPlex assay and Simultaneous Dual Emission detection describes the use of the PHERAstar^® FSX microplate reader for detecting dual emission AlphaLISA^® assays. In the study, an AlphaLISA simultaneous dual emission module was evaluated for its ability to perform two AlphaLISA assays at the same time (Emission A 615 nm; Emission B 545 nm) (Fig. 2). A Z prime value of 0.863 was obtained based on data for this module for protein at 256 μg/ml as positive control and no protein as a negative control. The assay was therefore determined to be suitable for high throughput analysis using simultaneous dual emission without any compromise in data quality.

dTAGs are a new variation of targeted protein degrader technology that can be used for target validation in high throughput screens. Like PROTACs, dTAGs are bivalent degraders that can induce the degradation of a target protein. However, the difference between dTAGs and PROTACs is that one of the ligands does not bind directly to an epitope specific for the target protein. Instead, this ligand binds to a dTAG-binding site artificially introduced into the target protein complex by genetic editing techniques like CRISPR (clustered regularly interspaced palindromic repeats)/Cas9. This refinement circumvents the need to identify and synthesize specific ligands for proteins of interest and allows a streamlined validation of potential target proteins by induced degradation. dTAGs are ideal tools for performing target validation studies for dose-dependent effects. In the application note dTAG Assay for Targeted Degradation of Proteins the Z prime values were determined for the dTAG-v1 degrader (Fig. 3). Values greater than 0.8 for different experiments confirmed the quality of these assays and their suitability for high throughput screening on 1536-well microplate formats.In the application note Real-time detection of Gs and Gi signaling in living cells G-protein coupled receptor signaling was detected using genetically encoded cADDis Upward and Downward biosensors from Montana Molecular. The assays were performed on BMG LABTECH’s CLARIOstar^® microplate reader and kinetic and dose response data were obtained for Gi and Gs signaling. These cell-based assays involved measurements using HEK293 cells performed in 96-well microplates. Each biosensor was demonstrated to have robust Z prime values (Fig. 4) for well characterized responses of HEK293 cells to isoproterenol.You can learn more about how the PHERAstar FSX and CLARIOstar can support a wide range of biological assays, including those that require consideration of Z statistics, in a video available here on the BMG LABTECH website. 

BMG LABTECH solutions

For high-throughput screening, BMG LABTECH’s PHERAstar^® FSX microplate reader offers high sensitivity, fast read times, and downscaling compatibility at a performance level suited to the best possible outcomes for the Z prime values of different biological assays.

The dedicated lasers of the PHERAstar FSX for excitation significantly improve assay performance and ensure lower limits of detection as they yield higher excitation energy at a specific wavelength. Simultaneous Dual Emission combined with paired photomultiplier tubes allow concomitant detection of dual emission assays, cutting reading times in half. In addition, they provide less variability and a broad dynamic range. This is particularly useful in dual emission fluorescence-based assays that need to be performed at high throughput, like TR-FRET or fluorescence polarization measurements. 

Since TR-FRET assays are robust and relatively easy to automate and miniaturize, they are widely used in drug and high-throughput screening applications. The main application of TR-FRET assays in high throughput screening is protein-protein or ligand-receptor interaction studies. The PHERAstar FSX delivers the outstanding performance needed to make highly sensitive TR-FRET measurements with low background noise for these types of assays. This in turn ensures superior Z prime values for most assays of this type.

On the PHERAstar FSX, measurements can take place without stopping plate movement, which significantly reduces read times for an entire microplate. Additional features include a wide range of flash numbers that can be tailored to assay requirements and a high data sampling rate for very fast reactions. The Enhanced Dynamic Range and auto Z-height focus features ensure that every plate is automatically measured with a setting that provides the best sensitivity and signal-to-blank ratios.

For lower throughput and assay development, both the VANTAstar^® and CLARIOstar Plus allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options. 

Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturized assays, short measurement times, and offer considerable savings on materials and other resources.

All BMG LABTECH readers come with a software package that includes the MARS data analysis interface. Here, Z statistics can be easily calculated and automated with the help of data reduction templates. Templates can be linked to specific detection protocols, enabling data generation and calculation of Z statistics with just two mouse clicks. Moreover, templates can be exported to be shared with other MARS users which helps to ensure the reliability of the analysis and the consistency of the results.

Whichever BMG LABTECH reader you choose Z prime values may help guide your assessment of the quality of your measurements for a particular assay and microplate reader set up. 

Conclusion

The demand for new research discoveries and translational efforts to develop new drugs will continue to rise as the life sciences continues to grow in the coming years. New opportunities in all areas of research, including high throughput screening, will benefit from the inclusion of microplate readers as part of solutions for increased speed and precision. The application of statistical parameters like Z prime value to this type of work environment will continue to deliver significant benefits to the life sciences when used in the right context and with due consideration of the relevance to different types of assays.
 
You can read more about Z prime values and other performance criteria for microplate readers in the BMG LABTECH eBook Microplates in action: Recommendations for use.

References

1. Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 1999;4(2):67-73. doi: 10.1177/108705719900400206.
2. Bar H, Zweifach A. Z' Does Not Need to Be > 0.5. SLAS Discov. 2020 Oct;25(9):1000-1008. doi: 10.1177/2472555220942764. Epub 2020 Aug 4. PMID: 32749188.
3. An WF, Tolliday N. Cell-based assays for high-throughput screening. Mol Biotechnol. 2010 Jun;45(2):180-6. doi: 10.1007/s12033-010-9251-z.