Get ready to avoid tedious ELISAs by using a robust and sensitive method
Polo-like kinase 1 (Plk1) is a well-established mitotic regulator involved in many biological functions throughout the cell cycle, including key checkpoints. Pre-clinical evidence suggests that the molecular targeting of Plk1 could be an effective therapeutic strategy in a wide range of cancers.The question still remains in the scientist’s mind: which assay to set up for an HTS screen?Only a biochemical assay format would be possible in that case. And, as a gold-standard technique, ELISA might appear to be the first option. However, it is certainly not at all suitable for HTS conditions.The answer is provided by Tae Gi Kim in this article. Step-by-step, he shows how to set up a TR-FRET binding assay. A side-by-side comparison with ELISA clearly shows both the robustness and the reliability of his assay. When developing or assessing an assay to test the effectiveness of various drugs, you always want to quantify how well the assay works. By measuring the Z’ factor, Tae Gi Kim was able to show higher assay reproducibility and signal stability compared to ELISA. This is confirmed through a pilot library screening of >1,000 natural compounds. Use an “add and read” technology makes it possible to run the screen in a time-effective manner with respect to conventional methods. This screen would have been a nightmare with ELISA.Consider it for your own protein-protein interaction assays and screenings.Simply build your own protein complex with biochemical reagents and run a robust assay to identify potent compounds. You’ll also save a significant amount of enzyme in the process.Perhaps the time has come to put your favorite ELISA on the shelf.
Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z’ factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z’ factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g] isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.
Biol. Pharm. Bull. 2017;40(9):1454-1462.