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.
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.
Ward
L D
LD
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Howlett
G J
GJ
Hammacher
A
A
Weinstock
J
J
Yasukawa
K
K
Simpson
R J
RJ
Winzor
D J
DJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018123:Receptors, Interleukin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D015850:Interleukin-6; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins