A direct comparison of THUNDER® assay performance with HTRF® and AlphaLISA™ SureFire® Ultra™ platforms

It’s no longer a secret that BioAuxilium’s THUNDER® is revolutionizing the market for drug discovery reagents by providing high-quality TR-FRET assay kits and reagents at affordable prices to the biomedical science community. The THUNDER® TR-FRET Cell Signaling Assay Kits are highly validated cell-based assay kits that enable the profiling of key cell signaling pathways. Recently, we have launched a panel of “High Performance” Phospho and Total STAT assays that exhibit improved sensitivity. In a new Application Note, we describe a head-to-head assessment of THUNDER®, HTRF®, and AlphaLISA™ SureFire® Ultra™ assays for the detection of phosphorylated STAT3 (Y705) in lysates from HeLa cells stimulated with interferon alpha-2b. The results show that the THUNDER High Performance Phospho-STAT3 assay outperformed HTRF and matched SureFire in terms of assay sensitivity while being simpler, faster, and more cost-effective.

Goal

We compared head-to-head the performance of THUNDER High Performance Phospho-STAT3 (Y705) assay in 384-well plate format with two other homogeneous assay platforms commonly used for drug discovery: HTRF and AlphaLISA SureFire Ultra.

Figure 1: THUNDER TR-FRET cell signaling assay principle

Methods

The three homogeneous assay platforms are based on the sandwich immunoassay principle. The assay principle of THUNDER is shown in Figure 1. All assays were conducted according to each manufacturer’s protocols using the standard two-plate transfer protocol for each kit, whereby the cells are seeded, treated and lysed in a 96-well culture plate, and lysates are then transferred to a low-volume 384-well assay plate for protein detection (see Figure 2). Performance metrics were signal-to-background (S/B) ratio, EC50, intra-assay variability (%CV), and stability of S/B ratio and EC50 value over time, ease of use, time-to-results and cost.

Figure 2: THUNDER assay workflow using the two-plate transfer protocol

Key Results

• The THUNDER assay surpassed the performance of the HTRF assay in terms of S/B ratios at all incubation points.
• The THUNDER assay generated a comparable S/B ratio to AlphaLISA SureFire but uses a shorter protocol and required half the time (1 hour versus 2 hours).
• In contrast to AlphaLISA SureFire, THUNDER is not sensitive to ambient light and normal temperature variations.
• THUNDER exhibited lower %CV compared to AlphaLISA SureFire.
• HTRF and SureFire are 68% and 77% more expensive than THUNDER.

Figure 3: Head-to-head assessment of three homogeneous Phospho-STAT3 (Y705) assays.

Conclusions

• All three phospho-STAT3 (Y705) assays produced robust concentration-responses curves for measuring the activation of STAT3 by IFNα2b in HeLa cell lysates.
• The THUNDER and AlphaLISA SureFire Ultra assays showed the highest and equivalent S/B ratios compared to the HTRF assay.
• However, THUNDER required fewer steps, was faster and had the lowest assay cost per well.

These key advantages, combined with rigorous assay validation using cell lysates from stimulated/inhibited cells, and higher flexibility in terms of kit sizes and formats (detection of either phosphorylated, total or phosphorylated plus total proteins with the same kit), make the THUNDER assay platform an alternative of choice for monitoring cellular protein phosphorylation.

See the Application Note for more information.