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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.

Type

Journal article

Journal

Current protocols in immunology / edited by John E. Coligan ... [et al.]

Publication Date

01/01/2002

Volume

Chapter 18