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. 2012 Jun 1;425(1):43-6.
doi: 10.1016/j.ab.2012.02.024. Epub 2012 Feb 28.

Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

Anna Y Kornilova  1 , Bethany Algayer, Michael Breslin, Victor Uebele
Affiliations

Development of a fluorescence polarization binding assay for asialoglycoprotein receptor

Anna Y Kornilova et al. Anal Biochem. .

Abstract

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.

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