Detection of Low‐Affinity Ligand‐Receptor Interactions at the Cell Surface with Fluorescent Microspheres

Marion H. Brown1

1 Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.2
DOI:  10.1002/0471142735.im1802s48
Online Posting Date:  May, 2002
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Abstract

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.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Detection of Cell‐Surface Ligands Using Soluble Chimeric Fusion Proteins Attached to Microspheres
  • Support Protocol 1: Biotinylation of MAb
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Cell‐Surface Ligands Using Soluble Chimeric Fusion Proteins Attached to Microspheres

  Materials
  • Avidin‐coated yellow fluorescent microspheres (0.46‐µm particles; Spherotech)
  • Biotinylated MAb specific for fusion partner (see protocol 2) in PBS/BSA/NaN 3: for rat CD4d3+4, use OX68 (European Collection of Animal Cell Cultures; Serotech)
  • PBS/BSA/NaN 3: PBS ( appendix 2A2) containing 0.125% (w/v) BSA and 0.02% (w/v) sodium azide (NaN 3)
  • Tissue culture supernatants (TCS) containing 2 µg/ml soluble recombinant fusion proteins, for both test proteins and a nonbinding negative control
  • Fusion protein–specific Fab fragments (optional) in PBS/BSA/NaN 3
  • Cells to be tested
  • 5 to 10 µg/ml cell surface–specific MAb (optional) in TCS
  • Water bath attached to a sonicator (e.g., ultrasonic liquid processor, Heat Systems–Ultrasonics)
  • Flat‐bottomed 96‐well culture plates
  • 12 × 75–mm round‐bottom tubes (Falcon)
  • Round‐bottomed 96‐well culture plates (optional)
  • Beckman GH‐3.7 rotor and GPR centrifuge with plate carriers, or equivalent
  • Additional reagents and equipment for counting cells and beads ( appendix 3A3) and for flow cytometry (see Chapter 5)

Support Protocol 1: Biotinylation of MAb

  Materials
  • 6‐(biotinamido)hexanoyl N‐hydroxysuccinimide ester (long‐chain biotin NHS ester, or LCB‐NHS; mol. wt. 456; Pierce; store desiccated at −20°C and warm to room temperature before opening)
  • Dimethylsulfoxide (DMSO)
  • 2 mg/ml (13 mM) purified MAb in PBS ( appendix 2A2)
  • 1 M Tris⋅Cl, pH 8 ( appendix 2A2)
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Figures

Videos

Literature Cited

Literature Cited
   Brown, M.H. and Barclay, A.N. 1994. Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis. Protein Eng. 7:515‐521.
   Brown, M.H., Preston, S., and Barclay, A.N. 1995. A sensitive assay for detecting low‐affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48. Eur. J.Immunol. 25:3222‐3228.
   Simmons, D. 1993. Cloning cell surface molecules by transient expression in mammalian cells. In Cellular Interactions in Development (D. Hartley, ed.) p. 93. IRL Press, Oxford.
   Sytkowski, A.J. 1990. Detection of cell surface components using fluorescent microspheres. Methods Enzymol. 184:353‐356.
   Tomschy, A., Fauser, C., Landwehr, R., and Engel, J. 1996. Homophilic adhesion of E‐cadherin occurs by a co‐operative two‐step interaction of N‐terminal domains. EMBO J. 15:3507‐3514.
   van der Merwe, P.A. and Barclay, A.N. 1994. Transient inter‐cellular adhesion—the importance of weak protein‐protein interactions. Trends Biochem. Sci. 19:354‐358.
Key References
   Brown et al., 1995. See above.
  A comprehensive paper that describes all points in this unit, and includes flow cytometry profiles of cells bound by covaspheres coated with CD4d3+4–immunoglobulin Fc fusion proteins. It also includes references to other studies using microspheres to detect ligands.
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