Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

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• 作者：Anna Y. Kornilova^a ; ^{anna_kornilova@merck.com} ; Bethany Algayer^a ; Michael Breslin^b ; Victor Uebele^a
• 关键词：Binding assay ; Membrane extracts ; Crude receptor extract
• 刊名：Analytical Biochemistry
• 出版年：2012
• 出版时间：1 June, 2012
• 年：2012
• 卷：425
• 期：1
• 页码：43-46
• 全文大小：414 K

文摘

Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z?factor of 0.73 was achieved in a 96-well format.