Fluorescence polarization assays (FP assays) are used in a variety of ways. From uncovering molecules in solution, to monitoring drug levels in clinical settings, and are enabling in drug discovery. These binding assays have been used to gain knowledge of molecular interactions, enzymatic activity, and nucleic acid hybridization. Moreover, scientists use FP to study small molecule-protein, antigen-antibody, and hormone-receptor binding.2
Once an FP immunoassay is developed, they are relatively straightforward to use. Because of the ability to rapidly detect the molecule of interest and simple mix-and-read homogenous qualities, the technique is a great fit for high throughput screening (HTS). Fluorescent polarization assays can use small amounts of assay reagents and can be miniaturized to 384-well, 1536-well formats, and beyond.
The Concept Behind Fluorescence Polarization Assays
The assay relies on the concept of fluorescence anisotropy to detect target molecules. Polarization decreases with elevated temperatures but rises as the viscosity and molecular weight increase. (That is why it’s important to equilibrate the assay reagents to the same temperature, to eliminate temperature changes to FP). Smaller molecules rotate more; therefore, not as likely to emit light within the polarized plane of excitation long enough to be measured.3 Since larger molecules move slower in solution, the FP technique uses this to its advantage.1,2,3
Antibody-tracer complex. Since the tracer is bound to the antibody it rotates slowly. Polarized light can then be detected because it is still within the same plane.
The assay detects the antibody-tracer complex in solution. If the target of interest displaces the tracer during competitive binding, then the fluorescence signal is lower due to the tracer speeding up because it is no longer bound to the much heavier antibody. The lower the concentration of the target molecule, the more tracer bound antibody can be detected as it is larger than the antibody alone. 1,2,3
Displaced tracer rotates at a much faster speed thus lowering the polarized light detected within the same plane.
Since the assay technique measures the size of the fluorescent molecule bound to target vs. the fluorescence intensity (FI), FP is better suited for molecule interaction studies.
Transcreener FP Assays
The Transcreener® ADP2 FP Assay exploits this concept to detect any enzyme that converts ATP to ADP. BellBrook Labs offers competitive FP immunoassays for the direct detection of not only ADP, but AMP, GMP, UDP, and GDP as well. This FP assay is ideal for drug discovery since the assay is simple, stable, and homogenous.
The assay has been used to identify inhibitors of methionyl-tRNA synthetase (MetRS) and RecA protein, which could lead to reducing specific disease and assist in the resistance to antibiotics.1 Its also been used to study a variety of kinases, GTPases, PDEs, and glycosyltransferases. These are just a few examples. FP will continue to be used to discover novel compounds that have therapeutic potential, and accelerate drug discovery.
- Hall, M. D., Yasgar, A., Peryea, T., Braisted, J. C., Jadhav, A., & Coussens, N. P. (2017). Fluorescence polarization assays in high-throughput screening and drug discovery: a review, 4(2), 1–41. https://doi.org/10.1088/2050-6120/4/2/022001.
- Manuscript, A. (2012). NIH Public Access, 6(1), 17–32. https://doi.org/10.1517/17460441.2011.537322. Fluorescence Polarization Assays in Small Molecule Screening
- Principle, H., See, A., & Perrin, F. (1926). Fluorescence polarization immunoassay.