Method for measuring dissociation rate coefficient by surface plasmon resonance analysis
View Patent ↗An object of the present invention is to provide a method for measuring the dissociation rate coefficient (Kd) by surface plasmon resonance analysis without measuring the theoretical maximum amount of binding (Rmax). The present invention provides a method for measuring the dissociation rate coefficient (Kd) of the reaction between an analyte molecule immobilized on a metal surface and a molecule that interacts with the analyte molecule by assaying changes in surface plasmon resonance signals using a surface plasmon resonance measurement device, wherein the signal and the slope of the dissociation curve of the surface plasmon resonance signal curves, or the signal ratio are used to calculate the dissociation rate coefficient (Kd).
1. A method for measuring the dissociation rate constant (Kd) of the reaction between an analyte molecule immobilized on a metal surface and a molecule that interacts with the analyte molecule by assaying changes in surface plasmon resonance signals using a surface plasmon resonance measurement device, comprising the steps of:
i. assaying changes in the surface plasmon resonance signals using a surface plasmon resonance measurement device; and,
ii. calculating the dissociation rate coefficient (Kd) using the signal and the slope of the dissociation curve of the surface plasmon resonance signal curves, or the signal ratio,
wherein the dissociation rate coefficient (Kd) is calculated based on the dissociation curve of the surface plasmon resonance signal curves using any of the following equations:
kd
=
-
R
′
(
t
)
R
(
t
)
≈
-
{
R
(
t
+
Δ
t
)
-
R
(
t
)
}
Δ
t
×
1
R
(
t
)
(
3
)
kd
=
1
Δ
t
Log
R
(
t
)
R
(
t
+
Δ
t
)
(
the
log
base
is
''
e
''
)
(
3
′
)
wherein t represents a time; and R(t+Δt) and R(t) represent an amount of binding at the time points t+Δt and t, respectively; and,
wherein changes in the surface plasmon resonance signal are measured using a surface plasmon resonance measurement device comprising a flow channel system having a cell formed on a metal film and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected on the metal film, in a state where the flow of the liquid has been stopped, after the liquid contained in the above flow channel system has been exchanged from a reference liquid containing no test substance to be measured to a sample liquid containing a test substance to be measured.
2. The method of claim 1 wherein a surface plasmon resonance measurement device which comprises a dielectric block, a metal film formed on one side of the dielectric block, a light source for generating a light beam, an optical system for allowing the above light beam to enter the above dielectric block so that total reflection conditions can be obtained at the interface between the dielectric block and the metal film and so that various incidence angles can be included, a flow channel system comprising a cell formed on the above metal film, and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of the light beam totally reflected at the above surface is used.