Development of a fluorescent ligand-binding assay using the AcroWell filter plate

J Biomol Screen. 2000 Dec;5(6):455-61. doi: 10.1177/108705710000500608.

Abstract

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.

Publication types

  • Comparative Study

MeSH terms

  • Cell Line
  • Combinatorial Chemistry Techniques
  • Drug Evaluation, Preclinical / instrumentation*
  • Europium
  • Fluorescence
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / metabolism*
  • Galanin / metabolism
  • Humans
  • Iodine Radioisotopes
  • Ligands
  • Radioligand Assay
  • Receptors, Galanin
  • Receptors, Neuropeptide / analysis
  • Receptors, Neuropeptide / metabolism

Substances

  • Fluorescent Dyes
  • Iodine Radioisotopes
  • Ligands
  • Receptors, Galanin
  • Receptors, Neuropeptide
  • Europium
  • Galanin