Binding of Small Peptides to Immobilized Antibodies: Kinetic Analysis by Surface Plasmon Resonance

David Andreu1, Paula Gomes2

1 Universitat Pompeu Fabra, Barcelona, 2 Centro de Investigação em Química da Universidade do Porto, Porto
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.9
DOI:  10.1002/0471142735.im1809s50
Online Posting Date:  November, 2002
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This unit describes a method for screening small viral peptides as specific antigens using a surface plasmon resonance (SPR) biosensor. The basic protocol in this unit is suited for direct single‐step SPR analysis of small ligand‐large receptor interactions, where small peptides are used as analytes (injected in the continuous buffer flow) and monoclonal antibodies (MAbs) are immobilized on the SPR sensor chip surface. An alternate protocol is included for situations where kinetic analysis is not possible and uses a surface competition assay to indirectly measure the kinetics of small analyte binding.

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Table of Contents

  • Basic Protocol 1: Direct Assay for Kinetics of Small Peptide Binding to Immobilized Monoclonal Antibodies Using an SPR Biosensor
  • Alternate Protocol 1: Alternative Kinetic Analysis of Small Peptide/large Antigen Competition on the Antibody Surface
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Direct Assay for Kinetics of Small Peptide Binding to Immobilized Monoclonal Antibodies Using an SPR Biosensor

  • recipeHBS‐EP running buffer (BIACORE BR‐1001‐88; 6 × 200 ml; also see recipe)
  • recipeMonoclonal antibody solutions (see recipe)
  • recipeImmobilization buffers (see recipe)
  • Amine coupling kit (BIACORE BR‐1000‐50, for 50 immobilizations):
  •  750 mg N‐ethyl‐N′‐(3‐dimethylaminopropyl)carbodiimide (EDC)
  •  115 mg N‐hydroxysuccinimide (NHS)
  •  10.5 ml ethanolamine hydrochloride
  • recipeRegenerating solutions for MAb surface (see recipe), e.g., 50 mM HCl or 10 mM NaOH
  • BIAnormalizing solution (BIACORE BR‐1003‐22, 90 ml): for normalization of BIACORE probe signal
  • recipePeptide solutions (see recipe)
  • Scrambled peptide (i.e., same mol. wt. and amino acid composition as peptide of interest, but with randomized sequence) as negative control (prepare as described in recipe for peptide solutions)
  • Personal computer running Microsoft Windows '95, '98, 2000 or NT
  • BIACORE 1000 SPR biosensor system including:
  •  BIACORE control 3.1 software
  •  CM5 sensor chips, certified grade (BIACORE BR‐1000‐12; three‐chip pack): carboxymethylated dextran matrix, with ≥ 4000 RU binding capacity for a 40 ‐kDa protein standard; user‐defined binding specificity.
  •  BIAevaluation 3.0 software

Alternate Protocol 1: Alternative Kinetic Analysis of Small Peptide/large Antigen Competition on the Antibody Surface

  • recipeHigh‐molecular‐weight competitor (HMWC) solutions (see recipe)
  • recipePeptide–HMWC mixtures (see recipe)
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Literature Cited

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Key References
   Fägerstam et al., 1992. See above.
  All of the above are original papers on the fundamentals, applications, limitations and strategies for real‐time biospecific interaction analysis using SPR biosensors.
   Hall et al., 1996. See above.
  The above are review papers on SPR biosensor technology for real‐time biospecific interacion analysis.
   Karlsson, 1994. See above.
  The three publications above are key sources on biosensor technology, from the manufacturer BIOSENSOR AB (Uppsala, Sweden), including instrumentation, software, and application manuals.
   Löfås, S. and Johnsson, B. 1990. A novel hydrogel matrix on gold surface in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J. Chem. Soc. Chem. Comm. 1526‐1528.
   Morton et al., 1995. See above.
   O'Shannessy and Winzor, 1996. See above.
   O'Shannessy et al., 1992. See above.
   O'Shannessy, D.J., Brigham‐Burke, M., Soneson, K.K., Hensley, P., and Brooks, I. 1993. Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: Use of non‐linear least squares methods. Anal. Biochem. 212:457‐468.
   Garland, P.B. 1996. Optical evanescent wave methods for the study of biomolecular interactions. Quart. Rev. Biophys. 29:91:117.
   Schuck, 1997. See above.
   Homola et al., 1999. See above.
   BIAcore Instrument Handbook. 1994. Pharmacia Biosensor AB, Uppsala, Sweden.
   BIAevaluation Software Handbook: version 3.0, 1997. BIAcore AB, Uppsala, Sweden.
   BIApplications Handbook, 1994. Pharmacia Biosensor AB, Uppsala, Sweden.
Internet Resources
  Web site of BIAcore.
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