Ligand-Peroxidase Conjugates for Quantification of Receptor-Mediated Transport into Cells

Jie      Chen; Amandio      Vieira

doi:10.2174/1386207311316040005

Abstract

Receptor-mediated cellular uptake of physiological regulators such as nutritional and hormonal factors represents a transport event with important consequences for cell differentiation, death, or proliferation. Although internalization pathways are important points of regulation, they have not been extensively explored as pharmacological targets in most cell types. An experimental strategy based on ligand-enzyme conjugates is presented in this report that may facilitate high-throughput screening for potent chemical modulators of the transport events. The method was tested on a human epidermoid cell line using a streptavidin-peroxidase conjugate, and human holo-transferrin as the model ligand, in a biotin-ligand conjugate. The proposed screening method is rapid, can be performed using multi-well plates, and involves small assay volumes. The modular nature of the ligand complex makes this method adaptable to the use of other biotinylated ligands, and the use of avidins conjugated to other enzymes. As is discussed, the method may also be applicable to other in vitro and in vivo transport assays.

Keywords: Cell transport, epidermoid cells, ligand conjugates, receptor-mediated endocytosis, screening assay, Endocytosis, clathrin-mediated endocytosis, transferrin endocytosis, transferrin RME model, ligand-enzyme conjugate assay.