Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface.
A capturing assay was used to monitor a Fab-antigen interaction using a BIACORE optical biosensor. The antigen, a truncated single-site mutant (F43V) version of the CD4 receptor, was captured onto the sensor surface using an immobilized nonneutralizing monoclonal antibody. While this assay design created an oriented antigen surface, the antigen slowly dissociated during subsequent binding of the Fab, thus complicating the binding responses. In this paper, we illustrate how binding events occurring on a decaying surface can be accurately described by globally fitting the response data to a model that accounts for the background surface decay. Support for the method was obtained by showing the equilibrium dissociation constant calculated from the kinetic rate constants (Kd = 2.20 +/- 0.01 nM) was similar to the value measured in solution using titration calorimetry (Kd = 2.6 +/- 0.5 nM). The ability to interpret rate constants from decaying surfaces significantly extends the types of experimental systems that can be quantitatively studied on optical biosensors.
Joss
L
L
Huntsman Cancer Institute, University of Utah, 15 N. 2030 E. Rm 2100, Salt Lake City, Utah, 84112-5330, USA.
Morton
T A
TA
Doyle
M L
ML
Myszka
D G
DG
D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D015704:CD4 Antigens / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007700:Kinetics; D055095:Optics and Photonics; D013499:Surface Properties; D013816:Thermodynamics
D015704:CD4 Antigens; D007140:Immunoglobulin Fab Fragments