Using Biacore to Measure the Binding Kinetics of an Antibody‐Antigen Interaction

Michael Murphy1, Laure Jason‐Moller1, JoAnne Bruno1

1 Biacore, Inc., Piscataway, New Jersey
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.14
DOI:  10.1002/0471142301.ps1914s45
Online Posting Date:  September, 2006
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Abstract

The optical phenomenon of surface plasmon resonance (SPR) used by Biacore systems enables the detection and measurement of protein‐protein interactions in real time, without the use of labels. In this unit, the application of Biacore technology to measure a protein‐protein interaction is described using an antibody and its antigen as an example. The affinity of the antibody for its antigen is determined by measuring the binding kinetics of the interaction. The protocols are divided into three major steps that are required for measuring binding kinetics using Biacore: (1) surface preparation, (2) assay development, and (3) kinetic analysis.

Keywords: Biacore; surface plasmon resonance (SPR); biosensor; label‐free; protein interaction analysis; kinetics; antibody

     
 
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Table of Contents

  • Basic Protocol 1: Identification of Optimal Immobilization pH (pH Scouting)
  • Basic Protocol 2: Immobilization of Ligand to Sensor Chip Using Amine Coupling
  • Basic Protocol 3: Regeneration Scouting: Identifying a Suitable Buffer for Surface Regeneration
  • Basic Protocol 4: Surface Performance Test to Assess Ligand Stability Under Regeneration Conditions
  • Basic Protocol 5: Kinetic Analysis for Ligand Immobilized Directly to Surface
  • Alternate Protocol 1: Immobilization of Ligand to Sensor Chip Using Ligand Capture Method
  • Alternate Protocol 2: Kinetic Analysis for Ligand Capture Method of Binding Ligand to Surface
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of Optimal Immobilization pH (pH Scouting)

  Materials
  • 200 ml HBS‐P+ (see recipe)
  • 1 mg/ml anti‐β2µ‐globulin antibody (Biacore)
  • 10 mM sodium acetate, pH 4.0, 4.5, 5.0, and 5.5
  • 50 mM NaOH
  • Biacore 3000, 2000, or T100 system
  • Sensor Chip CM5
  • 0.8‐ or 1.5‐ml polypropylene tubes with rubber caps (Biacore)

Basic Protocol 2: Immobilization of Ligand to Sensor Chip Using Amine Coupling

  Materials
  • 100 mM N‐hydroxysuccinimide (NHS)
  • 400 mM N‐ethyl‐N‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC)
  • 10 µg/ml anti‐β2µ‐globulin antibody in 10 mM sodium acetate, pH 5.5
  • 1 M ethanolamine
  • 50 mM NaOH
  • Biacore 3000, 2000, or T100 system
  • 0.8‐ml tubes
  • Sensor chip CM5

Basic Protocol 3: Regeneration Scouting: Identifying a Suitable Buffer for Surface Regeneration

  Materials
  • 32 nM β2µ‐globulin in HBS‐P+ (see recipe for HBS‐P+)
  • 10 mM glycine, pH 1.5, 2.0, 2.5, and 3.0
  • Biacore 3000, 2000, or T100 system

Basic Protocol 4: Surface Performance Test to Assess Ligand Stability Under Regeneration Conditions

  Materials
  • 32 nM β2µ‐globulin
  • 10 mM glycine, pH 2.5
  • Biacore 3000, 2000, or T100 system

Basic Protocol 5: Kinetic Analysis for Ligand Immobilized Directly to Surface

  Materials
  • 100 µg/ml (8.5 µM) β2µ‐globulin
  • HBS‐P+ (see recipe)
  • 10 mM glycine, pH 2.5
  • Biacore 3000, 2000, or T100 system

Alternate Protocol 1: Immobilization of Ligand to Sensor Chip Using Ligand Capture Method

  Materials
  • 100 mM N‐hydroxysuccinimide (NHS)
  • 400 mM N‐ethyl‐N‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC)
  • 25 µg/ml anti‐mouse IgG antibody in 10 mM sodium acetate, pH 5.0 (Biacore)
  • 1 M ethanolamine
  • 5 µg/ml solution of anti‐β2µ‐globulin in HBS‐P+
  • 10 mM glycine, pH 1.7
  • Biacore 3000, 2000, or T100 system
  • 0.8‐ml tubes
  • Sensor Chip CM5

Alternate Protocol 2: Kinetic Analysis for Ligand Capture Method of Binding Ligand to Surface

  Materials
  • 8.5 µM stock β2µ‐globulin (100 µg/ml)
  • HBS‐P+ (see recipe)
  • 10 mM glycine, pH 1.7
  • 5 µg/ml solution of anti‐β2µ‐globulin in HBS‐P+
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Figures

Videos

Literature Cited

   Andersson, K., Hamalainen, M., and Malmqvist, M. 1999. Identification and optimization of regeneration conditions for affinity‐based biosensor assays. A multivariate cocktail approach. Anal. Chem. 71:2475‐2481.
Key References
   Andersson et al., 1999. See above.
  This report describes a systematic approach to producing regeneration solutions and identifying regeneration conditions.
   Karlsson, R. and Fält, A. 1997. Experimental design for kinetic analysis of protein‐protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200:121‐133.
  This classic paper describes the suitability of using a dextran hydrogel on the sensor chip surface, describes the importance of using a reference, and explores the use of control experiments to gain understanding of reaction mechanisms.
   Myszka, D.G. 1999. Improving biosensor analysis. J. Mol. Recognit. 12:1‐6.
  This paper underscores the importance of careful experimental design when measuring binding kinetics with biosensors.
   Önell, A. and Andersson, K. 2005. Kinetic determinations of molecular interactions using Biacore: Minimum data requirements for efficient experimental design. J. Mol. Recognit. 18:307‐317.
  This work provides recommendations for the design of cost‐effective assays that deliver reliable kinetic measurements.
   Rich, R.L. and Myszka, D.G. 2005. Survey of the year 2003 commercial optical biosensor literature. J. Mol. Recognit. 18:1‐39.
  These literature surveys are most highly recommended as guides to careful experimental design, data analysis and presentation, and as excellent resources for outstanding biosensor publications.
   Rich, R.L. and Myszka, D.G. 2005. Survey of the year 2004 commercial optical biosensor literature. J. Mol. Recognit. 18:431‐478.
Internet Resources
   http://www.biacore.com/lifesciences/products/catalog/index.html
  Biacore catalog.
   https://www.biacore.com/lifesciences/service/online_support/irdb/index.html
  Immobilization and regeneration database.
   http://www.biacore.com/lifesciences/service/training/courses/usa_canada/biacorebasics/index.html
  Training courses.
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