Use of a Biosensor with Surface Plasmon Resonance Detection for the Determination of Binding Constants: Measurement of Interleukin-6 Binding to the Soluble Interleukin-6 Receptor
- Resource Type
- Authors
- Geoffrey J. Howlett; Richard J. Simpson; Larry D. Ward; Janet Weinstock; Annet Hammacher; Donald J. Winzor; Kiyoshi Yasukawa
- Source
- Biochemistry. 34:2901-2907
- Subject
- Recombinant Fusion Proteins
Molecular Sequence Data
Kinetics
Biosensing Techniques
In Vitro Techniques
Biochemistry
Chromatography, Affinity
Affinity chromatography
Escherichia coli
Humans
Amino Acid Sequence
Binding site
Surface plasmon resonance
Receptor
Binding Sites
Interleukin-6
Chemistry
Receptors, Interleukin
Receptors, Interleukin-6
Binding constant
Molecular Weight
Solubility
Covalent bond
Biophysics
Biosensor
- Language
- ISSN
- 1520-4995
0006-2960
The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection. First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor. A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6. A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.