Measurement of the Kinetics of Biomolecular Interactions Using the IAsys Resonant Mirror Biosensor

Andrew J. T. George1

1 Imperial College School of Medicine Hammersmith Hospital, London
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.5
DOI:  10.1002/0471142735.im1805s33
Online Posting Date:  May, 2001
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Abstract

In this unit, the resonant mirror biosensor or IAsys, produced by Affinity Sensors, is discussed for the measurement of the kinetics of biomolecular interactions. All biosensors operate using a common principle‐‐that binding events occurring on the sensing surface of the biosensor are converted by a transducer into an electronic signal which can then be processed by a computer to yield information on the amount of binding. Where biosensors vary is in their configuration and the nature of the transducer. Both the IAsys and BIACORE series of machines use optical techniques to detect the binding event, but the IAsys uses a resonant mirror device as the transducer, while the BIACORE uses surface plasmon resonance.When using biosensors the experiment can be divided into various phases, which are separately in this unit. The phases involve (1) the immobilization of the ligand onto the sensing surface; (2) addition of the ligate to the sensing surface, and following its association to the ligand, including the kinetics of binding; (3) washing of free ligate from chamber and following the dissociation of the ligand‐ligate complex; (4) regeneration of the surface; repetition of phases 2 and 3; and (5) data analysis. A protocol is also included for optimization of ligand pH for immobilization to the carboxymethylated dextran surfaces described in this unit.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immobilization of Ligands to Carboxymethylated Dextran Hydrogel Surfaces Using EDC/NHS Chemistry
  • Support Protocol 1: Optimization of pH for Immobilization of Ligands to Carboxymethylated Dextran Hydrogel Surfaces
  • Alternate Protocol 1: Immobilization of Ligands onto Aminosilane Surface Via Glutaraldehyde
  • Support Protocol 2: Determination of the Kinetics of Binding of Biomolecules to Ligands Using IAsys
  • Support Protocol 3: Regeneration of IAsys Ligand Binding Surfaces
  • Support Protocol 4: Analysis of IAsys Data Using Fastfit Software
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immobilization of Ligands to Carboxymethylated Dextran Hydrogel Surfaces Using EDC/NHS Chemistry

  Materials
  • PBS‐Tween: phosphate‐buffered saline (PBS; appendix 2A2) containing 0.05% (v/v) Tween 20
  • recipeNHS coupling kit (IAsys NHS‐2005; Affinity Sensors), with aliquots prepared according to the manufacturer's instructions (also see recipe)
  • Ligand of interest at as high a concentration as possible
  • 1 M ethanolamine, pH 8.5
  • 100 mM acetate buffer of appropriate pH (see protocol 2)
  • IAsys resonant mirror biosensor system (Affinity Sensors; see )
  • IAsys CM‐Dextran cuvettes (Affinity Sensors; FCD‐0101 for manual machine; FCD5101 for Auto+; see )

Support Protocol 1: Optimization of pH for Immobilization of Ligands to Carboxymethylated Dextran Hydrogel Surfaces

  Materials
  • PBS‐Tween: phosphate‐buffered saline (PBS; appendix 2A2) containing 0.05% (v/v) Tween 20
  • 100 mM sodium acetate buffers at pH 3, 3.5, 4, 4.5, 5, 5.5, and 6
  • Ligand at as high a concentration as available
  • IAsys resonant mirror biosensor system (Affinity Sensors; see )
  • IAsys CM‐Dextran cuvettes (Affinity Sensors; FCD‐0101 for manual machine, FCD5101 for Auto+; see )

Alternate Protocol 1: Immobilization of Ligands onto Aminosilane Surface Via Glutaraldehyde

  Materials
  • 5% (v/v) glutaraldehyde (prepare using 25% aqueous solution purchased from Sigma)
  • 0.1 M NaOH
  • 0.1 M HCl
  • 10 mM sodium phosphate, pH 7.7
  • 100 µg/ml ligand in 10 mM sodium phosphate, pH 7.7
  • 10 mg/ml bovine serum albumin (BSA) in 10 mM sodium phosphate, pH 7.7
  • Aminosilane cuvettes (Affinity Sensors; FCA‐0201 for manual machine; FCA‐5201 for Auto+; see )
  • IAsys resonant mirror biosensor system (Affinity Sensors; see )

Support Protocol 2: Determination of the Kinetics of Binding of Biomolecules to Ligands Using IAsys

  Materials
  • PBS‐Tween: phosphate‐buffered saline (PBS; appendix 2A2) containing 0.05% (v/v) Tween 20
  • Ligate
  • Cuvette with immobilized ligand (see protocol 1 or protocol 3)
  • Additional reagents and equipment for regeneration of the binding surface of the cuvette (see protocol 5) and analysis of data (see protocol 6)
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Figures

Videos

Literature Cited

   Buckle, P.E., Davies, R.J., Kinning, T., Yeung, D., Edwards, P.R., Pollard‐Knight, D., and Lowe, C.R. 1993. The resonant mirror: A novel optical biosensor for direct sensing of biomolecular interactions. Part II: Applications. Biosens. Bioelectronics 8:355‐368.
   Davies, R.J. and Pollard‐Knight, D. 1993. An optical biosensor system for molecular interaction studies. Am. Biotechnol. Lab. 11:52‐54.
   Edwards, P.R., Gill, A., Pollard‐Knight, D.V., Hoare, M., Buckle, P.E., Lowe, P.A., and Leatherbarrow, R.L. 1995. Kinetics of protein‐protein interactions at the surface of an optical biosensor. Anal. Biochem. 231:210‐217.
   George, A.J.T., French, R.R., andGlennie, M.J. 1995a. Measurement of kinetic binding constants of a panel of anti‐saporin antibodies using a resonant mirror biosensor. J. Immunol. Methods 183:51‐63.
   George, A.J.T., Jamar, F., Tai, M.‐S., Heelan, B.T., Adams, G.P., McCartney, J.E., Houston, L.L., Weiner, L.M., Opermann, H., Peters, A.M., and Huston, J.S. 1995b. Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: Technetium‐99m coordination by single‐chain Fv antibody fusion proteins through a C‐terminal cysteinyl peptide. Proc. Natl. Acad. Sci. U.S.A. 92:8358‐836.
   George, A.J.T., Rashid, M., and Gallop, J.L. 1997. Kinetics of biomolecular interactions. Expert Opinion on Therapeutic Patents 7:947‐963
   Karlsson, R. 1994. Real‐time competitive kinetic analysis of interactions between low‐molecular‐weight ligands in solution and surface‐immobilized receptors. Anal. Biochem. 221:142‐151
Internet Resources
   http://www.affinity‐sensors.com/
  Affinity sensors Web site.
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