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Development of a Fluorescent Ligand-Binding Assay Using the AcroWell Filter Plate

Pharnacopeia, Inc., Princeton, NJ, Bioscience Department, Purdue Pharma, Princeton, NJ 08540

Wendy Miller

Pharnacopeia, Inc., Princeton, NJ, Bioscience Department, Purdue Pharma, Princeton, NJ 08540

Jared N. Kravitz

Bioscience Department, Purdue Pharma, Princeton, NJ 08540

Philippe Samama

Pharnacopeia, Inc., Princeton, NJ, Department of Metabolic Diseases, Hoffman-La Roche, Inc., Nutley, NJ 07110

Dan Fitzpatrick

Pharnacopeia, Inc., Princeton, NJ, Amgen, Thousand Oaks, CA

Kevin Seeley

Pall Gelman Laboratory, Ann Arbor, MI

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.

Journal of Biomolecular Screening, Vol. 5, No. 6, 455-461 (2000)
DOI: 10.1177/108705710000500608


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