U.S. patent application number 17/731009 was filed with the patent office on 2022-08-11 for method for determining the interaction between a ligand and a receptor.
The applicant listed for this patent is INSINGULO AB. Invention is credited to Fredrik Hook, Tim Kaminski.
Application Number | 20220252589 17/731009 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220252589 |
Kind Code |
A1 |
Kaminski; Tim ; et
al. |
August 11, 2022 |
METHOD FOR DETERMINING THE INTERACTION BETWEEN A LIGAND AND A
RECEPTOR
Abstract
Described is a sample holder assembly including a functionalized
test well wall, which may be used in combination with a light
source.
Inventors: |
Kaminski; Tim; (Billdal,
SE) ; Hook; Fredrik; (Alingsas, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSINGULO AB |
Molndal |
|
SE |
|
|
Appl. No.: |
17/731009 |
Filed: |
April 27, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17263582 |
Jan 27, 2021 |
11346839 |
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PCT/EP2019/070925 |
Aug 2, 2019 |
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17731009 |
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International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 21/64 20060101 G01N021/64; G01N 33/573 20060101
G01N033/573 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2018 |
EP |
18187241.7 |
Claims
1.-27. (canceled)
28. A sample holder assembly configured to be used in combination
with microscopy, comprising: a sample holder plate comprising a
plurality of bottomless test wells, a bottom plate attached to said
sample holder plate via a material, thereby forming a well bottom
wall of said plurality of bottomless test wells, said material
having a refractive index (N.sub.a) that is lower than a refractive
index (N.sub.g) of said bottom plate.
29. The sample holder assembly according to claim 28, wherein the
well bottom wall is functionalized with a second ligand which faces
the interior of a bottomless test well of the plurality of
bottomless test wells whereby the second ligand is immobilized.
30. The sample holder assembly according to claim 28, wherein said
bottom plate comprises glass.
31. The sample holder assembly according to claim 29, wherein the
second ligand is a pharmaceutical drug.
32. The sample holder assembly according to claim 31, wherein the
second ligand is melagatran.
33. The sample holder assembly according to claim 28, wherein said
material is UV curable and/or resistant to buffer solutions.
34. The sample holder assembly according to claim 28, wherein said
material is an adhesive.
35. The sample holder assembly according to claim 28, which
comprises a microtiter plate.
36. The sample holder assembly according to claim 28, wherein the
sample holder assembly is combined with a light source configured
to provide a light beam into the well bottom wall such that the
light beam propagates throughout the entire well bottom wall
thereby creating an evanescent field in the plurality of bottomless
test wells.
37. The sample holding assembly according to claim 28, wherein the
microscopy is selected from the group consisting of image analysis,
Surface Plasmon Resonance (SPR), Total Internal reflection
Fluorescence (TIRF), waveguide imaging, interferometric scattering,
light filed microscopy, epi fluorescent microscopy, laser scanning
microscopy, orbital scanning microscopy, local enhancement
microscopy, structured illumination microscopy, RESOLF microscopy,
spatially illuminated illumination, omnipresent localization
microscopy, and x-ray microscopy.
38. The sample holding assembly according to claim 28, wherein the
microscopy is TIRF microscopy.
39. The sample holding assembly according to claim 36, wherein the
light source is a TIRF source.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a method for determining
the interaction between a first ligand such as a test compound and
a receptor such as a target molecule. The present disclosure also
relates to a sample holder assembly for use in said method.
BACKGROUND
[0002] Biosensors are used in drug discovery for the generation of
binding kinetic data, which can be used to further the
understanding of structure-kinetic relationships during the
compound optimization process. Kinetic information is frequently
deduced through microfluidics-based biosensor platforms like
surface plasmon resonance (SPR), as the optimized fluidics and the
high sampling rates allow for an exact description of molecular
association and dissociation processes. Information about extended
dissociation processes can eventually provide possibilities to
enhance compound efficacy and safety and thus may help to ensure
therapeutic success if correlated with the corresponding
pharmacokinetic features.
[0003] All optical biosensor platforms follow the same guiding
principle by attaching one interaction partner, usually the drug
target protein, oligonucleotide or even entire cells, to a
biosensor surface. The modified surface is subsequently challenged
with solutions containing test compounds in order to obtain direct
binding information or to study the biological consequences of
binding when working with cellular systems. This assay
configuration is called a direct binding assay (DBA).
[0004] Surface plasmon resonance (SPR) and optical waveguide (OWG)
make use of an evanescent-wave phenomenon and thus are able to
measure changes in the refractive index that are proportional to
changes in molecular mass at the sensor surface. In contrast, bio
layer interferometry (BLI) operates through the analysis of
interference patterns that enables to monitor changes in the
effective optical thickness of the layer that is in direct contact
with the sensor. Common for all platforms is the capability for
time resolved measurements of the binding interaction, particularly
when using microfluidics-based systems. As optical biosensor
systems do not require any labelling of the used reagents, they are
often referred to as label-free technologies aiming to reduce the
number of assay artefacts that may possibly be introduced by
labelling either the target or the compound.
[0005] One common property of microfluidic based biosensor
platforms is the need for two separate experiments. First, the
sensor surface is brought into contact with the analyte. Thereby, a
reaction, the rate of which being, in the simplest case, a
convolution of the association, k.sub.on, and dissociation,
k.sub.off, rate constants, is measured. Typically, this reaction is
observed until equilibrium coverage on the sensor surface is
reached, the coverage of which being determined by the equilibrium
dissociation constant, K.sub.d=k.sub.off/k.sub.on. Subsequently,
the sensor surface is exposed to an analyte-free solution with the
intention to monitor the dissociation reaction alone, from which
k.sub.off can be directly deduced.
[0006] This type of biosensing comes with a number of drawbacks,
especially if the target molecule is a membrane protein. It has
been estimated that about 60% of all approved drug targets are
membrane proteins. Especially for membrane proteins the success
rate is low .about.30%.
[0007] Many drug targets, especially membrane proteins are
incompatible with the immobilization at the sensor surface.
Therefore, time and resource intensive modifications has to be
introduced to the target.
[0008] They require a high density of the drug target at the
surface since the signal amplitude is proportional to the number of
immobilized targets.
[0009] These systems are only capable of detecting net changes at
the surface. They are therefore blind to the binding and unbinding
dynamics at equilibrium.
[0010] These sensor platforms offer very limited number of sensor
surfaces. SPR system usually have 3-4 separate sensor surfaces.
That means that if the target becomes dysfunctional, the sensor is
"lost". Next to the sensor cost the device cannot record any
further data for that particular surface. This limits the
throughput dramatically.
[0011] Before a new run can be started, the sensor surface has to
be free of any test compound. For long residence time compounds
this can be challenging and surface regeneration is often limiting
the throughput.
[0012] The sensitivity is limited by technical features of the
biosensor platform.
[0013] One way to mitigate the problem of target immobilization is
to keep the drug target in solution. Instead of immobilizing the
target a so-called tool-compound is immobilized, with which the
suspended target is known to interact with in a specific manner.
The test compound is added to the target outside the instrument to
allow target molecules and test compound molecules to react, and
the resulting solution is thereafter injected over the immobilized
tool compound. This technology is commonly referred to as
Inhibition in Solution Assay (ISA). Hence, by investigating how
different concentrations of a test compound influences the binding
of the target to the tool compound, one can determine the
equilibrium dissociation constant, K.sub.d, of the test compound to
the drug target. J. Med. Chem., (2013), 56, 3228-3234 discloses
this type of assay. For conventional label-free technologies, the
immobilized tool-compound has to dissociate from the drug target
slowly. Obtaining such a tool-compound is challenging.
[0014] In contrast to DBAs, the configuration of label-free
technologies does not enable measurements of binding kinetics in
combination with an ISA, i.e. the association rate constant
k.sub.on and the dissociation rate constant k.sub.off. Mol.
Pharmacol. (1984), 25, 1-9 discloses that non-label free
technologies allow to measure kinetics in a very limited range by
competition experiments, and also describes mathematically how the
binding kinetics is changed if two ligands are competing for
binding to the target. If the kinetics of one of the ligands is
known (ligand A) and this ligand is labelled which makes it
possible to distinguish it from the other ligand (ligand B)--the
binding kinetics of ligand B can be determined by recording the
binding kinetics of ligand A. Anal. Biochem. (1975), 468, 42-49
discloses that these assays need a labelled tool-compound that
binds the target with high affinity.
[0015] Anal. Chem. (2015), 87, 4100-4103 discloses a
single-molecule based ISA (SMM-ISA) that allows to perform
inhibition in solutions assays with high sensitivity. For this
method the target is not immobilized at the surface but is instead
immobilized in/at a suspended freely diffusing liposome that carry
fluorescent dyes in its lipid environment. On the surface a
tool-compound that can bind to the target is immobilized. The
modified surface is imaged with a total internal reflection
fluorescence (TIRF) microscope. TIRF generates an evanescent field
of excitation light which will only excite liposomes that are close
to the surface (couple of 100 nm). By keeping the concentration of
liposomes low, single-liposomes binding to and dissociating from
the surface can be imaged. In contrast to the conventional methods
described above, the high sensitivity of the single-molecule assay
allows the affinity of the tool-compound to the drug target to be
orders of magnitude lower. In addition, the concentration of the
target-containing liposome can be orders of magnitude lower than in
conventional ISA.
[0016] However, in the first reports on SMM-ISA it was only the
binding kinetics between the target and the surface-immobilized
tool compound that could be determined, and not the dynamic kinetic
parameters (the association, k.sub.on, and the dissociation,
k.sub.off, rate constants) of the crucial interaction between the
target and the test compounds. An additional drawback was the low
throughput and high amount of manual work that had to be done. This
limits cost efficiency, reproducibility and thus reliability,
therefore not fulfilling industry requirements.
[0017] Langmuir, 2015, 31 (39), pp 10774-10780 discloses use of
total internal reflection fluorescence microscopy to characterize
the kinetics between CXCR3, a GPCR involved in inflammatory
responses, and two of its chemokine ligands, CXCL10 and CXCL11.
Fluorescence labeling of the lipid membrane, rather than the
membrane protein itself, of GPCR-containing containing native
vesicles, and immobilization of the corresponding ligand on the
surface, enabled the determination of the dissociation constant
between the receptor and the ligand in solution using
single-molecule equilibrium-fluctuation analysis. The interaction
between the CXCR3 and the chemokine ligands CXCL10 and CXCL11 was
made under stagnant liquid conditions.
[0018] J. Am. Chem. Soc., 2011, 133, 14852-14855 discloses kinetics
of ligand binding to membrane receptors from equilibrium
fluctuation analysis of single binding events. Stagnant liquid
conditions were used and each association and dissociation event
was monitored over time by TIRF microscopy in a microwell
format.
[0019] It is an object of the present disclosure to overcome or at
least mitigate some of the problems described above. Further, an
object of the present disclosure is to provide advantages and/or
aspects not provided by hitherto known technique.
SUMMARY
[0020] The present disclosure provides a method for determining the
interaction between a first ligand and a receptor,
said method comprising a sequence of process steps: a) providing a
first solution free from the first ligand and comprising a
concentration C.sub.i of the receptor, b) contacting said first
solution with a test well wall functionalized with a second ligand
while recording the number of binding events between the receptor
and the second ligand during a time interval t.sub.1, and c) adding
a test solution free from said receptor and comprising a
concentration C.sub.n of the first ligand to the first solution
thereby providing a second solution while continuing recording the
number of binding events between the second ligand and the receptor
in said second solution during a time interval t.sub.2.
[0021] Importantly, the recording of the number of binding events
in the method described herein takes place during the addition of
the test solution comprising the first ligand, i.e. step c) and may
be started shortly after the addition of the first ligand before
the second solution reaches equilibrium. This is in contrast to
stagnant conditions where the recording of the binding events takes
place only after the addition of the first ligand and the binding
between the first ligand and the receptor has reached equilibrium
or quasi-equilibrium. Thus, the method described herein uses
non-stagnant conditions such as non-stagnant liquid conditions. As
a result, the determination of the interaction between the first
ligand and the receptor is made prior to equilibrium binding
between said first ligand and said receptor. It is a significant
advantage of the method described herein that it does not require
waiting for the first ligand and the receptor to reach or
substantially reach equilibrium which makes the total time for
making the measurement longer. Instead, the method described herein
allows for a fast throughput when measurements are performed. Of
course, this is particularly advantageous in screening
applications.
[0022] It will be appreciated that the recording of binding events
in step b) is optional. In an example, the recording of binding
events in step b) does not take place or takes place prior to, such
as just prior to, step c). Thus, there is provided a method as
described herein wherein step b) is:
b) contacting said first solution with a test well wall
functionalized with a second ligand while the number of binding
events between the receptor and the second ligand during a time
interval t.sub.1 is recorded or is not recorded.
[0023] A further advantage of the method described herein is that
the binding kinetics between the second ligand and the receptor do
not have to be known or determined.
[0024] The time interval t.sub.2 is at least 1/k.sub.obs of the
binding reaction between the first ligand and the receptor. Thus,
the time interval t.sub.2 may be equal to or above 1/k.sub.obs of
the binding reaction between the first ligand and the receptor.
k.sub.obs may be measured and calculated as described herein.
[0025] In an example, the recording of the binding events between
the second ligand and the receptor takes place until the first
ligand and the receptor have reached equilibrium binding. In a
further example, the recording of the binding events between the
second ligand and the receptor takes place until and after the
first ligand and the receptor have reached equilibrium binding.
[0026] It will be appreciated that the method described herein may
be performed in such a way that the recording of the binding events
between the receptor and the second ligand is not interrupted
between steps b) and c), i.e. the recording takes place in a
continuous manner. Further, the time interval t.sub.1 intends the
time range from adding the receptor in step b) until the first
ligand is added in step c). Moreover, the time interval t.sub.2
intends the time range from adding the first ligand in the test
solution in step c) until at least the first ligand and the
receptor have reached equilibrium binding as described herein or is
at least 1/k.sub.obs.
[0027] The equilibrium binding described herein may be binding at
quasi-equilibrium, i.e. near equilibrium or substantially at
equilibrium.
[0028] The method described herein may further comprise a step
d):
d) determining the interaction between the first ligand and the
receptor based on the binding events recorded in steps b) and
c).
[0029] In this document, the first ligand may be a test compound.
The terms "first ligand" and "test compound" may be used
interchangeably. Further, in this document the term "receptor" may
be a target such as a drug target. The terms "receptor" and
"target" may be used interchangeably. Moreover, in this document
the second ligand may be a tool compound. The terms "second ligand"
and "tool compound" may be used interchangeably.
[0030] The sequence of process steps of the method described herein
may be performed in full in each of a plurality of test wells.
[0031] Each method step may be carried out at the same time in each
test well, i.e. the first step may be carried out at the same time
in each test well and then each consecutive step may be carried out
at the same time in each test well. Alternatively, the method steps
may be carried out at different times in the test wells.
[0032] The number of receptors binding to the test wall may be
recorded before and after the addition of the first ligand. These
recorded binding events may be summed up for the plurality of test
wells. The number of binding events before addition of the first
ligand may be recorded during a time interval t.sub.1 as described
herein. The number of binding events after addition of the first
ligand may be recorded during a time interval t.sub.2 as described
herein. The time intervals t.sub.1 and t.sub.2, respectively, may
be the same or different. The summed up recorded binding events
before the addition of the test compound, and the summed up
recorded binding events after the addition of the test compound,
respectively, may subsequently be used for determining the
interaction between the first ligand and the receptor. When the
method steps are not carried out at the same time in each test well
this has to be taken into consideration to make a correct summing
up the binding events in the plurality of test wells.
[0033] Further, the sequence of process steps may be performed for
a number of different concentrations C.sub.n of the first ligand in
said test solution. Accordingly, the method described herein may
comprise a step e):
e) repeating step c) at an increasing concentration C.sub.n of the
first ligand in said test solution.
[0034] Importantly, the test solution of the method described
herein is free from receptor and is added to the first solution.
Thus, the first ligand and the receptor are not allowed to react
prior to adding the test solution to the first solution. These
features distinguish the method described herein from Inhibition in
Solution Assays (ISA) where the test solution comprises both a
receptor and a test compound which are allowed to react prior to
being added to a functionalized surface.
[0035] The test well wall described herein may be a test well
bottom wall of a single test well or a plurality of test wells. The
test well wall is functionalized with a second ligand which faces
the interior of the test wells whereby the second ligand is
immobilized. The functionalization of the test well wall may take
place using techniques known in the art. For instance, the test
well wall may be treated with a piranha solution followed by
functionalization of the test well wall.
[0036] The receptor may be used as such or used in combination with
a vehicle. The receptor and/or the vehicle may be labelled or
unlabelled. The labelling may include a fluorophore. In an example,
there is provided a vehicle comprising a fluorophore. The
combination of the receptor with a vehicle allows for immobilizing
said receptor without or substantially without negatively impacting
the receptor structure. Additionally, the vehicle can provide the
receptor an environment representing or mimicking its native
environment, especially if it is a membrane receptor. The
immobilized receptor may be soluble or substantially soluble in a
selected solvent or the vehicle or a combination of the vehicle and
the solvent.
[0037] Examples of vehicles that may be used for immobilizing the
receptor include, but are not limited to, at least one of the
following: a liposome, a liposome, a dendrimer, a dendrone, a
complexed lanthanide, a quantum dot, a nanodiamond, a lipid
disc.
[0038] The first ligand and the second ligand may be the same or
different. Thus, in contrast to methods requiring the dissociation
rate of the receptor from the surface bound second ligand to be
known or measured the first and second ligand may be different from
each other.
[0039] The receptor may be a pharmaceutical drug receptor. For
instance, the receptor may comprise or consist of thrombin.
Additionally or alternatively, the first ligand and/or the second
ligand may be a pharmaceutical drug. For instance, the
pharmaceutical drug may be melagatran.
[0040] The steps b) and/or c) of the method described herein may
comprise use of a microscope. Frequently, the use of a microscope
is appropriate for recording the number of binding events between
the receptor and the second ligand. Examples of suitable techniques
that may be used in conjunction with the method of the present
disclosure include image analysis, Surface Plasmon Resonance (SPR),
Total Internal Reflection Fluorescence (TIRF), waveguide imaging,
interferometric scattering, light field microscopy, epi
fluorescence microscopy, laser scanning microscopy, orbital
scanning microscopy, local enhancement microscopy, structured
illumination microscopy, RESOLFT microscopy, spatially modulated
illumination, omnipresent localization microscopy, and/or x-ray
microscopy.
[0041] The method described herein may allow for and/or comprise
determination for the first ligand at least one of, i.e. one or
more of, the following: an observed rate constant k.sub.obs, an
association rate constant k.sub.on, a dissociation rate constant
k.sub.off, an equilibrium dissociation constant K.sub.d, a
fractional occupancy.
[0042] The rate constant k.sub.obs characterizes how fast the
receptor becomes occupied with the first ligand. It depends on the
association rate constant k.sub.on, the dissociation rate k.sub.off
and the concentration C.sub.n of the test compound as shown in
equation 1.
k.sub.obs=k.sub.onC.sub.n+k.sub.off Equation 1:
[0043] The receptor bound by the first ligand cannot bind not
anymore to the second ligand or binds differently to the
immobilized second ligand. The number of binding events per unit
time is denominated .alpha.(t) and is given by equation 2
below.
.alpha.(t)=k.sub.on,VC.sub.i,free(t<t.sub.inh) Equation 2:
[0044] It will be appreciated that C.sub.i,free(t<t.sub.inh) is
the concentration of receptor not bound by the 10 first ligand or
the immobilized second ligand at a time t which is less than the
time at which the first ligand (inhibitor) is added, i.,e.
t.sub.inh. Further, k.sub.on,V is the association rate constant for
the first ligand to the receptor, said receptor binding to a
vehicle such as a vehicle.
[0045] Upon the addition of said first ligand with concentration
C.sub.n, C.sub.i,free(t<t.sub.inh) changes into C.sub.i,free(t)
as shown in equation 3 below.
C.sub.i,free(t)=C.sub.i,free(t<t.sub.inh)(1-.beta.(t)) Equation
3:
[0046] In equations 3 and 4, .beta.(t) represents the fraction of
receptors in complex with the first ligand at a time t. .beta.(t)
may have a value between 0 and 1, i.e. 0<.beta.(t)<1.
.beta.(t) may be determined as shown in equation 4 below.
.beta.(t)=C.sub.n/(C.sub.n+K.sub.d)*[1-exp(-k.sub.obs*(t-t.sub.inh)]
Equation 4:
[0047] As described herein, C.sub.nis the concentration of the
first ligand, K.sub.d is the equilibrium constant, k.sub.obs is the
observed rate constant, t is the time at which the measurement is
made and t.sub.inh is the time at which the first ligand
(inhibitor) is added.
[0048] The equilibrium dissociation constant K.sub.d for the first
ligand is described in Equation 5, where [L] is the concentration
of the first ligand, [R] is the concentration of the receptor and
[LR] is the concentration of the receptor binding to the test
compound.
K d = [ L ] .function. [ R ] [ L .times. R ] Equation .times.
.times. 5 ##EQU00001##
[0049] K.sub.d may also be expressed as the ratio between the
dissociation constant k.sub.off and the association constant
k.sub.on as shown in equation 6.
K.sub.d=k.sub.off/k.sub.on Equation 6:
[0050] The fractional occupancy is the amount of receptor-ligand
complex divided by the initial amount of receptor as shown in
Equation 7, where [Ligand Receptor] is the concentration of the
first ligand binding to the receptor at equilibrium and [Total
receptor] is the initial concentration of the receptor.
Fractional .times. .times. occupancy = [ Ligand .times. .times.
Receptor ] [ Total .times. .times. Receptor ] Equation .times.
.times. 7 ##EQU00002##
[0051] The fractional occupancy may also be expressed as indicated
in Equation 8, where [Ligand] is the concentration of free first
ligand at equilibrium of the binding reaction and K.sub.d is as
described herein.
Fractional .times. .times. occupancy = [ Ligand ] [ Ligand ] + K d
Equation .times. .times. 8 ##EQU00003##
[0052] When the method of the present invention is performed in a
plurality of test wells these may form part of a sample holder
assembly such as a microtiter plate. Thus, the sample holder
assembly may comprise or consist of a microtiter plate. Such sample
holder assemblies allow for performing a large number of
experiments in a time-and cost efficient way. Further, performing
the method in a plurality of test wells allows for increasing the
sensitivity since the number of recorded binding events in the test
wells may be summed up thereby providing more data points.
[0053] Advantageously, the use of a plurality of test wells as
described herein allows for avoiding so-called ligand depletion
which may occur when there are too many receptor binding spots for
too few ligands so that the ligands become depleted. In order to
avoid ligand depletion the receptor concentration should be
lowered. However, this leads to poor signal strength since the
signal strength in binding assays is usually proportional to the
concentration of the receptor. The use of a plurality of test wells
in the method described herein compensates for poor signal strength
by allowing for summing up data points from several test wells.
[0054] It has been found that TIRF is a suitable technique to be
used in conjunction with the method described herein. Accordingly,
there is provided a method as described herein wherein the sample
holder assembly is configured to be used in combination with Total
Internal Reflection Fluorescence (TIRF) microscopy and comprises:
[0055] a sample holder plate comprising a plurality of bottomless
test wells [0056] a bottom plate attached to said sample holder
plate by means of a material such as an attachment means such as an
adhesive thereby forming a well bottom wall of said plurality of
test wells, said material such as attachment means such as adhesive
having a refractive index N.sub.a that is lower than a refractive
index N.sub.g of said bottom plate.
[0057] Traditionally, the TIRF source is provided from below the
bottom plate of the sample holder assembly. This would be time
consuming for a plurality of wells since the TIRF source then would
have to be moved around. Instead, the TIRF source may be placed so
that the light beam is propagated throughout the entire bottom
plate. However, this also requires that the light beam does not
leak into e.g. the test well bottom wall. Additionally, the
attachment means such as an adhesive should be able to attach
reliably and fast to the walls of the sample holder assembly when
put together and also withstand chemicals such as solutions and
reagents added to the sample holder wells.
[0058] For instance, the attachment means may be an adhesive.
Advantageously, the adhesive described herein may be UV curable
and/or resistant to buffer solutions. The UV curability allows for
convenient and fast attachment of the functionalized test well wall
to the walls of the sample holder plate. The resistance to buffer
solutions prevents the functionalized test well wall from being
detached from the sample holder plate.
[0059] The adhesive may comprise a silane from at least one of the
following: alkylsilanes, aminosilanes, epoxysilanes, hydrosils,
mercaptosilanes, methacrylic silanes.
[0060] In order to allow for the light beam from the TIRF source to
propagate throughout the entire bottom plate without being leaked
into adjacent media such as the sample holder bottom plate the
adhesive should be selected to have a refractive index N.sub.a that
is lower than a refractive index N.sub.g of said bottom plate. In
this way, an evanescent wave will be created closely to an inner
surface of the functionalized test well wall. Fluorescent receptors
or fluorescent vehicles immobilizing the receptor, i.e.
fluorophores, will then become excited and fluoresce in proximity
of the said surface. In this way, the measured fluorescence will
originate only from the fluorophore in proximity of the surface
while fluorophores further away from the surface will not
fluoresce.
[0061] Accordingly, the present disclosure provides a method as
described herein, wherein the sample holder assembly is combined
with a TIRF source configured to provide a light beam into the well
bottom wall such that the light beam propagates throughout the
entire well bottom wall thereby creating an evanescent field in the
plurality of wells.
[0062] The sample holder assembly may be prepared in advance to
suit an intended application. For instance, the sample holder
assembly bottom plate may be prepared by surface modification for a
particular application and then attached to the remainder of the
sample holder assembly with the aid of the attachment means
described herein.
[0063] Further, the present disclosure provides a use of a method
as described herein for evaluating the binding kinetics between two
or more different first ligands and a receptor without varying the
second ligand.
[0064] The present disclosure also provides a sample holder
assembly configured to be used in combination with TIRF microscopy,
comprising: [0065] a sample holder plate comprising a plurality of
bottomless test wells, [0066] a bottom plate attached to said
sample holder plate by means of a material such as an attachment
means such as an adhesive thereby forming a well bottom wall of
said plurality of test wells, said adhesive having a refractive
index N.sub.a that is lower than a refractive index N.sub.g of said
bottom glass plate (3). The material such as an attachment means
such as an adhesive may be UV curable and/or resistant to buffer
solutions. The sample holder plate may comprise or consist of a
microtiter plate.
[0067] The sample holder assembly disclosed herein may be combined
with a TIRF source configured to provide a light beam into the well
bottom wall such that the light beam propagates throughout the
entire well bottom wall thereby creating an evanescent field in the
plurality of wells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 is a graph showing the number of binding events as a
function of time for liposome immobilized thrombin before and after
the addition of melagatran.
[0069] FIG. 2 is a graph showing a dose response curve for
melagatran added to liposome immobilized thrombin.
[0070] FIG. 3 is a graph showing the observed binding rate
k.sub.obs as a function of the concentration of an added melagatran
solution, and linear regression providing k.sub.on between
melagatran and thrombin.
[0071] FIG. 4 is a graph showing the number of binding events in a
single test well as a function of time for liposome immobilized
thrombin before and after the addition of melagatran.
[0072] FIG. 5 is a graph showing the number of binding events in a
two test wells as a function of time for liposome immobilized
thrombin before and after the addition of melagatran.
[0073] FIG. 6 is a graph showing the number of binding events in a
one hundred test wells as a function of time for liposome
immobilized thrombin before and after the addition of
melagatran.
[0074] FIG. 7 is a cross section view of a microtiter plate
comprising a plurality of test wells comprising a functionalized
bottom plate attached to said microtiter plate by means of an
adhesive.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0075] FIG. 7 shows a cross section of a sample holder assembly 100
comprising a sample holder plate 1 comprising a plurality of
bottomless test wells 2, a bottom plate 3 attached to the sample
holder plate 1 by means of an adhesive 4 thereby forming a well
bottom wall 5 of said plurality of wells 2. The adhesive has a
refractive index N.sub.a that is lower than the refractive index
N.sub.g of said bottom plate 3.
[0076] As explained herein, the adhesive 4 may be elected to allow
for a TIRF light beam to propagate throughout the entire bottom
plate without leaking into adjacent media such as the bottom plate.
Thereby an evanescent wave is created closely to the bottom plate
which may be used in the detection of binding events as described
herein.
[0077] The well bottom wall 5 may be functionalized with a tool
compound as described herein.
Further Items
[0078] The present disclosure provides the following items.
Item 1:
[0079] A method for determining the interaction between a first
ligand and a receptor,
said method comprising a sequence of process steps: a) providing a
first solution free from said first ligand and comprising a
concentration C.sub.i of the receptor, b) contacting said first
solution with a test well wall functionalized with a second ligand
while recording the number of binding events between the receptor
and the second ligand during a time interval t.sub.1, c) adding a
test solution free from said receptor and comprising a
concentration C.sub.n of the first ligand to said first solution
thereby providing a second solution while recording the number of
binding events between the second ligand and the receptor of said
second solution during a time interval t.sub.2.
Item 2:
[0080] A method according to item 1, wherein the sequence of
process steps is performed in full in each of a plurality of test
wells.
Item 3:
[0081] A method according to item 1 or 2, wherein the sequence of
process steps is performed for a number of different concentrations
C.sub.n of the first ligand in said test solution.
Item 4:
[0082] A method according to item 1 or 2 further comprising a step
d):
d) repeating step c) at an increasing concentration C.sub.n of the
first ligand in said test solution.
Item 5:
[0083] A method according to any one of the preceding items,
wherein
the receptor is combined with a vehicle such as a liposome, a
dendrimer, a dendrone, a complexed lanthanide, a quantum dot, a
nanodiamond or a lipid disc, thereby providing an immobilized
receptor.
Item 6:
[0084] A method according to any one of the preceding items,
wherein the vehicle comprises a fluorophore.
Item 7:
[0085] A method according to any one of the preceding items,
wherein the first ligand and the second ligand are the same or
different.
Item 8:
[0086] A method according to any one of the preceding items,
wherein:
the receptor is a pharmaceutical drug receptor, and/or the first
ligand and/or the second ligand is/are a pharmaceutical drug.
Item 9:
[0087] A method according to any one of the preceding items,
wherein step b) and/or step c) comprise(s) use of a microscope.
Item 10:
[0088] A method according to any one of the preceding items, which
comprises determination for the first ligand at least one of the
following: an observed rate constant k.sub.obs, an association
constant k.sub.on, a dissociation constant k.sub.off, an
equilibrium dissociation constant K.sub.d, a fractional
occupancy
Item 11:
[0089] A method according to any one of items 2-10, wherein the
plurality of test wells form part of a sample holder assembly such
as a microtiter plate.
Item 12:
[0090] A method according to item 11, wherein the sample holder
assembly is configured to be used in combination with Total
Internal Reflection Fluorescence (TIRF) microscopy, and comprises:
[0091] a sample holder plate (1) comprising a plurality of
bottomless test wells (2), [0092] a bottom plate (3) attached to
said sample holder plate (1) by means of an adhesive (4) thereby
forming a well bottom wall (5) of said plurality of wells (2), said
adhesive (4) having a refractive index (N.sub.a) that is lower than
a refractive index (N.sub.g) of said bottom plate (3).
Item 13:
[0093] A method according to any one of items 10-12, wherein said
adhesive (4) is UV curable and/or resistant to buffer
solutions.
Item 14:
[0094] A sample holder assembly (10) configured to be used in
combination with TIRF microscopy, comprising: [0095] a sample
holder plate (1) comprising a plurality of bottomless test wells
(2), [0096] a bottom plate (3) attached to said sample holder plate
(1) by means of an adhesive (4) thereby forming a well bottom wall
(5) of said plurality of wells (2), said adhesive (4) having a
refractive index (N.sub.a) that is lower than a refractive index
(N.sub.g) of said bottom glass plate (3).
Item 15:
[0097] A method according to item 11 or a sample holder assembly
(10) according to claim 14, wherein the sample holder assembly (10)
is combined with a TIRF source configured to provide a light beam
into the well bottom wall (5) such that the light beam propagates
throughout the entire well bottom wall (5) thereby creating an
evanescent field in the plurality of wells (2).
EXAMPLES
Abbreviations
[0098] CHES N-Cyclohexyl-2-aminoethanesulfonic acid [0099] HBS
Hepes buffered solution [0100] HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) [0101] LED
Light Emitting Diode [0102] NHS N-Hydroxysuccinimid [0103] .mu.l
microliter [0104] L liter [0105] LOD lower limit of detection
[0106] nm nanometer [0107] nM nanoMolar [0108] mg milligram(s)
[0109] ml milliliter [0110] mM millimolar [0111] PBS Phosphate
buffered soluions [0112] PEG4 Polyethylene glycol, i.e.
H--(O--CH.sub.2--CH.sub.2).sub.4--OH. [0113] PC Phosphatidylcholine
[0114] PEG polyethylene glycol [0115] PLL-g-PEG poly-L-lysine
grafted PEG [0116] RT room temperature [0117] sec second(s) [0118]
UV ultraviolet [0119] V/V volume percent
Materials and Methods
[0120] All lipids were bought from Avanti Polar Lipids.
[0121] PII-g-PEG (11354-X=200-2000-3.5%) and PLL-g-PEG-biotin
(11835-X=200-3400-3.5%) were bought from Nanosoft Polymers.
[0122] The thrombin binding peptide was synthezised upon customer
specification by ThermoFisher Scientific. Melagatran was purchased
from SantaCruz Biotechnology.
[0123] Dymax 3025 is a product of Dymax Corporation.
[0124] All other chemicals if not stated differently were bought
from Sigma. All chemicals were suitable for molecular biology
purposes.
Preparation of Liposomes:
[0125] To yield liposomes with a diameter of .about.100 nm first
the required lipids were solved in chloroform and mixed. In total 5
mg 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, 0.01 mg,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl
and 0.005 mg
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl) were mixed together. The lipid mixture was
vacuum dried overnight. The dried lipids were hydrated in 1 ml HBS
(150 mM NaCl, 20 mM/L HEPES at pH 7.2) under gentle agitation. The
lipid suspension was extruded through a PC-membrane with 100 nm
pore size eleven times. Concentration of the liposomes was
determined by light absorption at 544 nm and the concentration of
the liposome solution was adjusted to 2.5 mg/ml. This equals a
liposome concentration of approximately 30 nM.
Preparation of Protein:
[0126] To immobilize thrombin at the liposomes via click chemistry
an azide-group was introduced to thrombin via NHS coupling.
Therefore, 100 .mu.L of human-thrombin at 2 mg/ml was mixed with
200 .mu.L high salt PBS-buffer (10 mM Na.sub.2HPO.sub.4, 1.8 mM
KH.sub.2PO.sub.4, 400 mM NaCl+33.5% Glycerol (v/v) at pH 7.4 and 13
.mu.L NHS-PEG4-Azide at 10 mM. The mixture was incubated for 30
minutes at RT. The reaction was stopped by the addition of 500
.mu.L high salt PBS-buffer (10 mM Na.sub.2HPO.sub.4, 1.8 mM
KH.sub.2PO.sub.4, 400 mM NaCl+33.5% Glycerol (v/v) at pH 6.6.
Immobilization of Protein:
[0127] 1.5 .mu.L of the thrombin-azide were mixed with 48.5 .mu.L
icecold CHES-buffer (20 mM CHES, 150 mM NaCl) at pH 8.5 and
subsequently 50 .mu.L of the liposome solution is added. This mix
was stored for at least 30 minutes on ice. 10 .mu.L of the reaction
mix was diluted with 990 .mu.L icecold CHES-buffer (20 mM CHES, 150
mM NaCl) at pH 8.5.
Preperation of Surfaces:
[0128] A glass plate with 0.17 mm thickness was incubated in base
piranha solution at 373 Kelvin for 30 minutes. The cleaned glass
plate was rinsed with water and dried. An UV curable adhesive
(Dymax 3025) was supplemented with .about.1%(v/v)
(3-Aminopropyl)-triethoxysilane. The adhesive mixture was thinly
spread at the lower side of the bottomless microplate. The glass
plate was positioned on top of the bottomless microplate so it
formed the bottom. After the adhesive was spread fully, it was
cured according to manufacturer instructions. After curing, into
each well of the microplate 10 .mu.L of a solution containing 1
mg/ml PLL-g-PEG and 1 mg/ml PLL-g-PEG-biotin are added. The plate
was incubated for at least 1 h under gentle orbital agitation.
After the incubation each well was washed 10-times with HBS-buffer.
The dilution ratio of every washing step was at least 1:10. After
washing, into each well 10 .mu.L of a solution containing 100
.mu.g/mL neutravidin was added. This was incubated for at least 4 h
under gentle orbital agitation. All wells were washed as previously
described and 10 .mu.L of a solution containing 10 ug/ml a thrombin
binding peptide linked to biotin
(GVGPRSFKLPGLA-Aib-SGFK-PEG.sub.4-biotin) was added to all wells.
The microplate was incubated at least for 1 h under gentle orbital
agitation. The peptide was the tool compound to bind the thrombin
immobilized at the vesicles.
[0129] Finally the microplate was washed as previously described
and the residual buffer volume in each well is 30 .mu.L.
[0130] The Microscope Setup:
[0131] The single molecule microscope was based on a Nikon Ti-E
base. As light source for epifluorescence a LED-white light source
is used. The Objective was a 60x APO TIRF objective. Images are
recorded via a HAMAMATSU Orca-FLASH 4.0V2 sCMOS camera. The sample
stage was motorized and quipped with a microwell holder.
[0132] On top of the microscope liquid handling robotics were
installed (Andrew, Andrew Alliance).
Melagatran Dilution Series:
[0133] An 8 times 1:3 dilution series of melagatran starting at 1.6
uM/L. The buffer for the dilution series was CHES-buffer (20 mM
CHES, 150 mM NaCl) at pH 9.5.
Example 1
[0134] The microwell plate comprising 384 wells was placed in the
microwell plate holder at the microscope. Subsequently the
measurement was started and conducted fully automatically.
[0135] The single molecule measurement included the following steps
in each well: [0136] 1. The appropriate microwell prepared as
indicated above was placed over the objective and the objective was
adjusted so its focal plane is placed at the inner surface of
microwell. [0137] 2. The well was washed with 70 .mu.L CHES-buffer
(20 mM/L CHES, 150 mM/L NaCl) at pH 9.5. [0138] 3. 5 .mu.L of a
solution containing CHES buffer and the liposome onto which
thrombin was attached was added to the well and the well content
mixed. [0139] 4. The acquisition of time lapse movie with 901
images and an acquisition rate of 10 sec.sup.-1 was started [0140]
5. After 20 seconds of acquisition 5 .mu.L solution containing the
appropriate concentration of melagatran and CHES buffer was added
and the well content mixed rapidly (<0.5 sec). [0141] 6. The
acquisition of image data was continued till 901 frames were
recorded.
[0142] Steps 1-6 were repeated three times for each intended
concentration of melagatran. Eight concentrations of melagatran
were tested, namely 200 nM, 66.7 nM, 22.2 nM, 7,4 nM, 2.5 nM, 0.8
nM 0.3 nM and 0.1 nM. For each tested concentration of melagatran
steps 1-6 above were performed in full in each well. Each method
step was carried out at the same time in each well, i.e. step 1 was
carried out at the same time in each well and then each consecutive
step was carried out at the same time in each well.
[0143] The recorded image data was analysed with the aim to
determine the number of new bound liposome in each well. In a first
step all objects that are similar in shape to a reference object in
each well were detected.
[0144] In a second step it was determined which objects were bound
to the surface. As indication that an object was bound to the
surface its mobility was analysed. If the mobility was below a
threshold value (the object has not moved more than a pixel (here
215-304 nm between two consecutive frames) the object was
considered as immobile and therefore bound to the surface. The
number of bound liposomes, i.e. the number of binding events, was
recorded. The number of binding events for all wells were summed up
before and after the addition of melagatran, respectively, to
provide a cumulative number of binding events. This was done for
each concentration of melagatran.
[0145] The cumulative number of bound liposomes before and after
the addition of melagatran was plotted versus time as shown in FIG.
1. In FIG. A the concentration of melagatran was 200 nM and the
concentration of thrombin was 15 pM. In FIG. 1 t.sub.inh is the
time when the solution of melagatran was added to the solution
containing the liposome immobilized thrombin, and cumsum on-events
[#] is the number of recorded binding events.
[0146] Before the test compound melagatran was added the binding
rate of liposomes to the surface was observed to be constant over
time. Plotting the cumulative number of binding events versus time
turned out to be a linear function where the slope equals the
binding rate. During the injection and mixing of the test compound
the binding rate to the surface was increased. After a short
equilibration time the binding rate was normalized again. The data
acquired during this mixing period was not used for analysis. The
cumulative number of binding events was analysed as described
herein and the observed binding rate constant k.sub.obs was
extracted. Once the binding of melagatran to thrombin had reached
its equilibrium the cumulative number of binding events versus time
was increasing linearly again as shown in FIG. 1.
[0147] The ratio of the initial slope and the final slope was
calculated. This was repeated three times for each concentration of
melagatran. This ratio equals the fractional occupancy of thrombin
by melagatran at the respective concentration. The equilibrium
dissociation constant K.sub.d was then calculated from the equation
below, wherein [melagatran].sub.0 is the concentration of the added
melagatran.
Fractional .times. .times. occupancy = [ m .times. e .times. l
.times. a .times. g .times. a .times. t .times. r .times. a .times.
n ] 0 [ m .times. e .times. l .times. a .times. g .times. a .times.
t .times. r .times. a .times. n ] 0 + K d ##EQU00004##
The equilibrium dissociation constant K.sub.d was provided from a
dose response curve wherein the fractional occupancy was plotted
versus the added concentration of melagatran as shown in FIG. 2,
which gave a K.sub.d value of about 3 nM.
[0148] FIG. 3 shows the observed binding rate constant k.sub.obs
plotted as a function of the concentration of the added test
compound melagatran. Linear regression of the observed rate
k.sub.obs versus the concentration of melagatran allowed for
calculation of the association rate k.sub.on between melagatran and
thrombin, which was found to be 21 .mu.M.sup.-1s.sup.-1.
[0149] Since the equilibrium dissociation constant K.sub.d equals
the dissociation rate k.sub.off divided by the association rate
k.sub.on, it was possible to calculate k.sub.off by myltiplying the
k.sub.on value of 21 .mu.M.sup.-1s.sup.-1 with the K.sub.d value of
3 nM thereby providing a k.sub.off value of about 0.06
s.sup.-1.
[0150] It was concluded that the method described herein allows for
determining k.sub.obs, k.sub.on, k.sub.off, and K.sub.d for a test
compound and also the fractional occupancy of a receptor by the
test compound. Thus, the method described herein allows for
determining the interaction kinetics between a test compound and a
receptor.
Example 2
[0151] In this example, the method steps described in Example 1
were performed in full in a single well, in each of two wells and
in each of 100 wells. The thrombin concentration was 1 pM. The
melagatran concentration was 7.4 nM.
[0152] First, an experiment was performed in a single well. The
number of binding events was plotted as a function of time as shown
in FIG. 4. Due to the low number of binding events the observed
rate constant k.sub.obs could not be reliably fitted. Therefore,
the number of binding events were collected and summed up before
and after the addition of melagatran, respectively, for two wells.
The result is shown in FIG. 5, and it was found that k.sub.obs
could be fitted to provide a k.sub.obs value of 0.43 sec.sup.-1. A
further experiment was performed in analogy with the two well
experiment but instead of two wells one hundred wells were used.
The result is shown in FIG. 6, and it was found that k.sub.obs
could be fitted to provide a k.sub.obs value of 0.22 sec.sup.-1. In
FIGS. 4,5 and 6 t.sub.inh is the time when the solution of
melagatran was added to the solution containing the liposome
immobilized thrombin, and cumsum on-events [#] is the number of
recorded binding events.
[0153] It was concluded that the sensitivity of the method
described herein is enhanced by performing the method steps in a
plurality of wells and summing up the recorded number of binding
events of the wells before and after the addition of the test
compound.
[0154] It was also concluded that the method described herein
allows for reliable measurement of the observed rate constant
k.sub.obs for a low receptor concentration such as a receptor
concentration that is lower than the lowest concentration that
corresponds to the LOD of ensemble averaging methods defining (i)
how much material of the receptor that is needed to operate the
assay and (ii) the tight binding regime with respect to high
affinity test compounds.
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