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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.

Original publication

DOI

10.1002/0471142735.im1802s48

Type

Journal article

Journal

Curr Protoc Immunol

Publication Date

05/2002

Volume

Chapter 18

Pages

Unit - 18.2

Keywords

Antibodies, Monoclonal, Biotinylation, Flow Cytometry, Fluorescence, Ligands, Microspheres, Receptors, Cell Surface, Recombinant Fusion Proteins