Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

• Published in: Analytical biochemistry 2012-06, Vol.425 (1), p.43-46
• Main Authors: Kornilova, Anna Y., Algayer, Bethany, Breslin, Michael, Uebele, Victor
• Format: Article
• Language: English
• Subjects: Animals; Asialoglycoprotein Receptor - analysis; Asialoglycoprotein Receptor - chemistry; Binding assay; binding capacity; Binding Sites; Crude receptor extract; Fluorescence; Fluorescence Polarization - methods; glycopeptides; hepatocytes; Kinetics; liver; Liver - metabolism; Membrane extracts; Rats; Rats, Sprague-Dawley
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2012.02.024

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.