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with relatively few preparation steps (Price, 2001).
Using antigens labelled with fluorescent markers,
FPI’s rely on the difference in anisotropy (or
polarisation) measurements of a labelled antigen
compared with the anisotropy of the labelled antigen
in association with an antibody (Terpetsching et al,
1994). More specifically, in FPI’s the emission
from the free labelled antigen must be depolarised.
Thus the rotational correlation time for the antigen
must be much shorter than that of the probe.
Therefore, when this method is applied to antigens
with a low molecular weight, fluorescein with a
lifetime near 4ns is typically chosen as the
fluorescent probe (Spencer et al, 1973). In
comparison with the free labelled antigen the
polarisation will increase when the labelled antigen
binds to the antibody.
In the case of GnRH-1, labelling demands either
the addition or removal of an amino acid. The
addition of an amino acid can lead to an alteration in
conformation due to the increase in size and change
in charge. This is less likely to occur if an amino
acid is removed. Therefore, in an attempt to preserve
the antibody binding site which is in the centre of
the peptide (Khan et al, 2003), we describe a method
that incorporates the 9-amino acid fragment of
GnRH-1 labelled with fluorescein to act as a
replacement for the conventional labelled antigen in
the assay. Furthermore, we use time-resolved
anisotropy techniques in an effort to reveal any
underlying issues that may arise from using the
labelled fragment in place of the labelled antigen.
Fluorescence spectroscopy is a technique that offers
sensitivity down to a single-molecule level, with a
signal-to-niose ratio that improves with
measurement duration, and is therefore well placed
for the development of ultrasensitive biosensors
such as fluorescence-based immunoassays.
2 THEORY
The timed-resolved fluorescence anisotropy, r(t), is
an angular correlation function that conveniently is
defined as (Lakowicz, 2006)
() ()
()
() 2 ()
vv vh
vv vh
tIt
rt
tIt
−
=
+
(1)
where I
vv
(t) and I
vh
(t) are the recorded fluorescence
intensities at time t following an excitation event at t
= 0, and where the first and second subscript refers
to the orientation of the excitation and emission
polariser respectively. In the simplest case, with a
fluorophore rigidly attached to a macromolecule that
undergoes free isotropic rotational diffusion, the
anisotropy is described by a mono-exponential
decay function
0
() exp
lobal
t
rt r
φ
⎛⎞
=−
⎜⎟
⎜⎟
⎝⎠
(2)
The time-zero ansiotrpoy r
0
can take the maximum
value 0.4 for one-photon excitation (assuming the
transition dipoles for absorption and emision are
parallel). The correlation time
φ
global
is related to the
hydrodynamic volume, V, of the macromoleule and
can be calculated form the Stokes-Einstein Debye
relationship
global
b
V
kT
φ
= (3)
wher
η
is the viscosity of the solvent, k
b
Boltzmann
constant and T the absolute temperature.
It is often observed when analysing the
fluorescence anisotropy recorded from fluorescently
labelled macromolecules, for example proteins, that
the anisotropy expression is given by a bi-
exponential decay law
12
() exp exp
local global
tt
rt b b
φφ
⎛⎞
⎛⎞
=−+−
⎜⎟
⎜⎟
⎜⎟
⎝⎠
⎝⎠
(4)
The correlation times are then often interpreted to
reflect on a local wobbling motion of the
fluorophore in its binding site,
φ
local
, and an overall
rotational diffusion of the macromolecule,
φ
global
. If
φ
global
>>
τ
, it will not be resolved in a time
correlated single-photon counting experiment, and
will only be evident by a plateau value of value r
∞
. If
there is also a fraction of free fluorophore that is not
bound to the macromolecule, as in our case, there
might be a fraction of the antigen that is not bound
to the antibody and then the anisotropy expression
reads
12
() exp exp
free local
tt
rt b b r
φφ
⎛⎞
⎛⎞
−+ − +
⎜⎟
⎜⎟
⎜⎟
⎝⎠
⎝⎠
(5)
In the present work anisotropy data was analysed
according to equation (5).
TOWARDS A NEW HOMOGENEOUS IMMUNOASSAY FOR GONADOTROPIN-RELEASING HORMONE BASED
ON TIME-RESOLVED FLUORESCENCE ANISOTROPY
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