Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology
| Autor: | Julia Mellentin-Michelotti, Pau-Miau Yuan, William E. Werner, Sheri Miraglia, Lolita Evangelista, Elana Swartzman |
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| Rok vydání: | 1999 |
| Předmět: |
Biophysics
Substance P Receptors Cell Surface Plasma protein binding CHO Cells Astrocytoma Ligands Transfection Biochemistry Binding Competitive chemistry.chemical_compound Cricetinae Tumor Cells Cultured Animals Humans Multiplex Galanin Receptor Molecular Biology Melanoma Ligand binding assay Cell Biology Receptors Neurokinin-1 Neuropeptide Y receptor Molecular biology Recombinant Proteins Receptors Neuropeptide Y chemistry Microscopy Fluorescence Neurokinin A Protein Binding |
| Zdroj: | Analytical biochemistry. 272(2) |
| ISSN: | 0003-2697 |
| Popis: | We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput. |
| Databáze: | OpenAIRE |
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