11
Surface Plasmon Resonance
Surface plasmon resonance (SPR) is a highly sensitive and precise method that
provides kinetic and affinity information. It's considered a gold standard, and has
many established standard protocols. Because of its quantitative aspects, SPR is
also used to help determine SARs of fragment analogs during downstream lead
optimization.
This method involves flowing solubilized analyte across a target protein that
has been immobilized to the surface of a sensor chip. Analyte binding increases
the mass immobilized on the chip; an event detectable by measuring changes
in the refractive index of light shone onto the chip. By testing different analyte
concentrations flowed over a constant amount of immobilized target, the k
on
, k
off
and K
d
of the interaction can be determined.
Optimization of the capture for each target is required and o en overlooked,
which may be the reason for lack of correlation with other orthogonal techniques.
For example, hit identification and subsequent lead optimization have different
requirements for capture density. During hit identification, one looks for high
density of captured protein whereas with lead optimization, one looks for the
opposite — less capture of target protein is desired.
Sensor chips can be reused by removing the analyte bound to the sensor chip. Not
to be overlooked, chip regeneration conditions must be optimized to completely
remove bound analyte while conserving the activity of the captured target protein.
Ideal for binding kinetics
and constants using label-
free detection
Immobilization of one
of the binding partners,
interference by DMSO
(a common solvent for
fragments) and regular
maintenance of its fluidics
components are known
obstacles
