Detection of low-affinity ligand-receptor interactions at the cell surface with fluorescent microspheres.
A method of producing highly avid multivalent ligand binding reagents for detecting low-affinity interactions at the cell surface is described in this unit. The principle is to immobilize multiple copies of extracellular regions of cell-surface molecules on plastic fluorescent beads, to present the coated beads to cells, and to analyze binding in a quantitative manner by flow cytometry. The method of attaching proteins to the beads is designed to maximize display of the ligand-binding region. The approach is applicable to immobilization of fusion proteins on a variety of beads, and can also be adapted for use with native proteins. A protocol for the biotinylation of MAbs is also presented.
Brown
Marion H
MH
Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, United Kingdom.
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D019921:Biotinylation; D005434:Flow Cytometry; D005453:Fluorescence; D008024:Ligands; D008863:Microspheres; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008024:Ligands; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins