Development of a fluorescent ligand-binding assay using the AcroWell filter plate.
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.
Valenzano
K J
KJ
Pharmacopeia, Inc., Princeton, NJ, USA.
Miller
W
W
Kravitz
J N
JN
Samama
P
P
Fitzpatrick
D
D
Seeley
K
K
D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D005063:Europium; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism*; D019004:Galanin / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D011869:Radioligand Assay; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide / Q000032:analysis / Q000378:metabolism
D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D008024:Ligands; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide; D005063:Europium; D019004:Galanin