U.S. patent application number 10/449823 was filed with the patent office on 2004-03-25 for method of coupling binding agents to a substrate surface.
This patent application is currently assigned to Biacore AB. Invention is credited to Safsten, Par, Tidare, Mattias.
Application Number | 20040058456 10/449823 |
Document ID | / |
Family ID | 29714428 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040058456 |
Kind Code |
A1 |
Safsten, Par ; et
al. |
March 25, 2004 |
Method of coupling binding agents to a substrate surface
Abstract
The present invention relates to a method of coupling multiple
binding agents to respective areas of a substrate surface by
hydrodynamic addressing, using two laminar fluid flows that flow
together in the same direction over the substrate surface with an
interface to each other to successively couple the binding agents
to the substrate areas, wherein each successive coupling of a
binding agent to a surface area is followed or preceded by
selective deactivation or activation of a selected surface area
according to a defined protocol. The invention also relates to the
use of such a binding agent-coupled substrate surface for
analytical purposes.
Inventors: |
Safsten, Par; (Uppsala,
SE) ; Tidare, Mattias; (Uppsala, SE) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Biacore AB
Uppsala
SE
|
Family ID: |
29714428 |
Appl. No.: |
10/449823 |
Filed: |
May 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60384626 |
May 31, 2002 |
|
|
|
Current U.S.
Class: |
436/518 ;
427/2.11 |
Current CPC
Class: |
B01J 2219/00511
20130101; B01J 2219/0059 20130101; B01J 2219/00626 20130101; B01J
2219/00605 20130101; B01J 2219/00596 20130101; B01J 2219/00689
20130101; B01J 2219/00617 20130101; B01J 2219/00286 20130101; C40B
40/10 20130101; B01L 3/502776 20130101; B01J 2219/00637 20130101;
B01J 2219/00657 20130101; B01L 2300/0877 20130101; B01J 2219/00677
20130101; B01J 2219/00353 20130101; B01J 2219/0074 20130101; B01J
2219/00527 20130101; B01J 2219/00585 20130101; B01J 2219/00704
20130101; B01J 19/0046 20130101; B01L 2200/0636 20130101; B01J
2219/00659 20130101; B01L 2300/0636 20130101; B82Y 30/00 20130101;
B01J 2219/00725 20130101 |
Class at
Publication: |
436/518 ;
427/002.11 |
International
Class: |
G01N 033/543; B05D
003/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2002 |
SE |
0201637-6 |
Claims
1. A method of coupling at least two different binding agents to
respective defined areas of a substrate surface by hydrodynamic
addressing based on two laminar fluid flows that flow together in
the same direction over the substrate surface with an interface to
each other, and selectively contacting a defined area of the
substrate surface with a desired fluid by positioning the interface
laterally through adjustment of the relative flow rates of the two
fluid flows, which method comprises at least one of hydrodynamic
addressing procedures A and B, wherein: procedure A comprises
immobilizing a first binding agent to a first area of the substrate
surface by contacting the area with a fluid containing the first
binding agent, deactivating the first area by subjecting the area
to a deactivating fluid, and immobilizing a second binding agent to
a second area of the substrate surface by contacting a substrate
surface area including the first and second areas with the second
binding agent; and procedure B comprises deactivating a first area
of the substrate surface by subjecting the area to a deactivating
fluid, immobilizing a first binding agent to a second area of the
substrate surface by contacting the first and second areas with a
fluid containing the first binding agent, activating at least a
part of the first area by subjecting the area to an activating
fluid, and immobilizing a second binding agent to the first area by
contacting the area with a fluid containing the second binding
agent.
2. The method according to claim 1, wherein procedure A comprises
the steps of: a) providing a substrate surface, at least part of
which is reactive to permit coupling of binding agents thereto; b)
passing over the substrate surface a laminar flow of a fluid
containing a first binding agent, and adjacent thereto a laminar
flow of a blocking fluid that does not interact with the substrate
surface, such that the two fluids flow together in the same
direction with an interface to each other, and adjusting the
relative flow rates of the two laminar fluid flows to position the
interface such that the first binding agent-containing fluid
selectively contacts a first reactive area of the substrate surface
to couple the first binding agent thereto; c) replacing the flow of
the first binding agent-containing fluid with a laminar flow of a
deactivating fluid, and positioning the interface laterally such
that the deactivating fluid selectively contacts at least the first
binding agent-coupled area but less than the whole reactive surface
area for deactivation thereof; and d) replacing the flow of the
deactivating fluid with a laminar flow of a fluid containing the
second binding agent, and positioning the interface laterally such
that the second binding agent-containing fluid selectively contacts
the deactivated area and an adjacent second reactive area of the
substrate surface to selectively couple the second binding agent to
the second area.
3. The method according to claim 2, which additionally comprises
the step of: e) replacing the flow of the second binding
agent-containing fluid with a laminar flow of deactivating fluid,
and positioning the interface laterally such that the deactivating
fluid selectively contacts at least the deactivated area and the
second binding agent-coupled area of the substrate surface for
deactivation thereof.
4. The method according to claim 3, wherein steps b) to e) are
repeated at least once to couple at least one additional binding
agent to a respective reactive area on the substrate surface.
5. The method according to claim 1, wherein procedure B comprises
the steps of: a) providing a substrate surface, at least part of
which is reactive to permit coupling of binding agents thereto; b)
passing over the substrate surface a laminar flow of a deactivating
fluid, and adjacent thereto a laminar flow of a blocking fluid that
does not interact with the substrate surface, such that the two
fluids flow together in the same direction with an interface to
each other, and adjusting the relative flow rates of the two
laminar fluid flows to position the interface such that the
deactivating fluid selectively contacts a first reactive area of
the substrate surface for deactivation thereof; c) replacing the
flow of the deactivating fluid with a laminar flow of a fluid
containing a first binding agent, and positioning the interface
laterally such that the first binding agent-containing fluid
selectively contacts the deactivated area and an adjacent second
reactive area of the substrate surface to selectively couple the
first binding agent to the second area; d) replacing the flow of
the first binding agent-containing fluid with a laminar flow of
activating fluid, and positioning the interface laterally such that
the activating fluid selectively contacts at least a part of the
deactivated first area for activation thereof; and e) replacing the
flow of the activating fluid with a laminar flow of a fluid
containing a second binding agent, and positioning the interface
laterally such that the second binding agent-containing fluid
selectively contacts the activated first area to selectively couple
the second binding agent thereto.
6. The method according to claim 5, wherein steps b) to e) are
repeated at least once to couple at least one additional binding
agent to a respective reactive area on the substrate surface.
7. The method according to claim 1, wherein at least one of the
reactive areas of the substrate surface comprises activated
functional groups.
8. The method according to claim 2, wherein step a) comprises
passing a laminar flow of activating fluid over at least a part of
the substrate surface to provide the at least partly reactive
surface.
9. The method according to claim 5, wherein step a) comprises
passing a laminar flow of activating fluid over at least a part of
the substrate surface to provide the at least partly reactive
surface.
10. The method according to claim 2, wherein step a) comprises
providing a surface with pre-activated functional groups.
11. The method according to claim 5, wherein step a) comprises
providing a surface with pre-activated functional groups.
12. The method according to claim 1, wherein at least one of the
reactive areas of the substrate surface comprises functional groups
capable of reacting with the binding agents without activation of
the functional groups.
13. The method according to claim 2, which comprises the step of,
prior to performing step b), passing over the substrate surface a
laminar flow of deactivating fluid and an adjacent laminar flow of
blocking fluid, and positioning the interface such that the
deactivating fluid selectively contacts a reactive edge area of the
substrate surface adjacent to the first area to be contacted with
binding agent in step b) for deactivation thereof.
14. The method according to claim 3, which comprises performing
steps b) to e) in the opposite lateral direction to the flow path
to couple at least one additional binding agent to a respective
reactive area of the substrate surface.
15. The method according to claim 14, which comprises activating an
additional part of the substrate surface prior to performing steps
b) to e) of claim 2 in the opposite lateral direction to the flow
path.
16. The method according to claim 3, wherein in steps c) and e) the
laminar flow of deactivating fluid is adjusted to contact also a
reactive area adjacent to the binding agent-coupled area to provide
a non-coupled area between neighbouring binding agent-coupled
areas.
17. The method according to claim 1, wherein procedure B comprises
immobilizing an additional binding agent to a third area situated
on the other side of the second area immobilized with the first
binding agent by selectively contacting the third area with a fluid
containing the additional binding agent.
18. The method according to claim 17, wherein the third area is
activated prior to immobilizing the additional binding agent.
19. The method according to claim 1, which comprises the steps of:
a) passing over the substrate surface a laminar flow of an
activating fluid, and adjacent thereto a laminar flow of a blocking
fluid that does not interact with the substrate surface, and
positioning the interface between the two fluids such that the
activating fluid selectively contacts a part of the substrate
surface for activation thereof; b) replacing the flow of activating
fluid with a laminar flow of blocking fluid, replacing the blocking
fluid with a laminar flow of a fluid containing a first binding
agent, and positioning the interface such that the fluid containing
the first binding agent selectively contacts the non-activated part
of the surface and an adjacent first area of the activated part of
the surface to couple the first binding agent to the first area; c)
replacing the flow of the first binding agent-containing fluid with
a laminar flow of blocking fluid, replacing the blocking fluid with
a laminar flow of a fluid containing a second binding agent, and
positioning the interface such that the binding agent-containing
fluid selectively contacts a second area of the activated part of
the surface adjacent to the first area coupled with the first
binding agent to couple the second binding agent to the second
area; d) replacing the flow of the second binding agent-containing
fluid with a laminar flow of a deactivating fluid, and positioning
the interface such that the deactivating fluid selectively contacts
the first and second areas coupled with the first and second
binding agents, respectively; e) replacing the flow of deactivating
fluid with a laminar flow of blocking fluid, replacing the flow of
blocking fluid with a laminar flow of activating fluid, and
positioning the interface such that the activating fluid
selectively contacts at least a portion of the non-activated part
of the surface, adjacent to the part activated in step a), for
activation thereof; and f) replacing the flow of blocking fluid
with a laminar flow of a fluid containing a third binding agent,
replacing the flow of activating fluid with a laminar flow of
blocking fluid, and positioning the interface such that the binding
agent-containing fluid selectively contacts the first and second
areas coupled with the first and second binding agents,
respectively, and a third area comprising at least a part of the
surface portion activated in step e) to couple the third binding
agent to the third area.
20. The method according to claim 19, which additionally comprises
the step of: g) replacing the flow of binding agent-containing
fluid with a laminar flow of a deactivating fluid, and positioning
the interface such that the deactivating fluid contacts at least
the area coupled with the third binding agent for deactivation
thereof.
21. The method according to claim 19, which comprises coupling a
fourth binding agent to a remaining activated area adjacent to the
third area after coupling of the third binding agent.
22. The method according to claim 1, which comprises the steps of:
a) passing over the substrate surface a laminar flow of a fluid
containing an activating fluid, and adjacent thereto a laminar flow
of a blocking fluid that does not interact with the substrate
surface, and positioning the interface between the two fluids such
that the activating fluid selectively contacts a part of the
substrate surface for activation thereof; b) replacing the flow of
activating fluid with a laminar flow of blocking fluid, replacing
the blocking fluid with a laminar flow of a fluid containing a
first binding agent, and positioning the interface such that the
fluid containing the first binding agent selectively contacts the
non-activated part of the surface and an adjacent first area of the
activated part of the surface to couple the first binding agent to
the first area; c) replacing the flow of the first binding
agent-containing fluid with a laminar flow of blocking fluid,
replacing the blocking fluid with a laminar flow of a fluid
containing a second binding agent, and positioning the interface
such that the binding agent-containing fluid selectively contacts a
second area of the activated part of the surface adjacent to the
first area coupled with the first binding agent to couple the
second binding agent to the second area; d) replacing the flow of
binding agent-containing fluid with a laminar flow of blocking
fluid, replacing the flow of blocking fluid with a laminar flow of
activating fluid, and positioning the interface such that the
activating fluid selectively contacts at least a portion of the
non-activated part of the surface, adjacent to the part activated
in step a), for activation thereof; and e) replacing the flow of
activating fluid with a laminar flow of a fluid containing a third
binding agent, and positioning the interface such that the binding
agent-containing fluid selectively contacts the area activated in
step d) to couple the third binding agent thereto.
23. The method according to claim 22, which additionally comprises
the step of: f) replacing the flow of binding agent-containing
fluid with deactivating fluid, and positioning the interface such
that the deactivating fluid contacts at least areas coupled with
the binding agents and previously not deactivated for deactivation
thereof.
24. The method according to claim 22, which comprises coupling a
fourth binding agent to a remaining activated area adjacent to the
third area after coupling of the third binding agent.
25. The method according to claim 22, which comprises deactivating
a part of the surface portion activated in step d), and after
coupling the third binding agent in step e), reactivating the
deactivated area and coupling a fourth binding agent thereto.
26. The method according to claim 1, wherein the substrate surface
is a sensing surface of a sensor.
27. The method according to claim 26, wherein the sensing surface
is provided in a flow cell.
28. The method according to claim 27, wherein the flow cell
comprises two inlets and one outlet.
29. A method of analysing a fluid sample for at least one analyte,
comprising sensitising a sensing surface with at least two
different binding agents according to the method of claim 1,
contacting the sensitised areas with the fluid sample, and
detecting interaction between the analyte and the sensing
surface.
30. A method of analysis, comprising sensitising a sensing surface
with at least two different binding agents according to the method
of claim 1, contacting the sensitised areas with at least one
analyte, and studying interaction between the analyte and the
sensing surface.
31. The method of claim 1, wherein a system comprising: a flow cell
having an inlet end and an outlet end, at least one sensing surface
on a wall surface within the flow cell located between the inlet
and outlet ends, wherein the flow cell has at least two inlet
openings at the inlet end and at least one outlet opening at the
outlet end, such that separate laminar flows entering the flow cell
through the respective inlet openings can flow side by side in the
same direction through the flow cell over the sensing surface,
means for applying laminar flows through the inlet openings, such
that the laminar flows pass side by side through the flow cell over
the sensing surface with an interface to each other that is
parallel to the direction of the laminar flows; and means for
varying the relative flow rates of the laminar fluids to displace
laterally the interface over the sensing surface; is used for for
performing the method.
32. A computer program product comprising program code means for
performing the steps of claim 1 when the program is run on a
computer.
33. A computer program product stored on a computer readable
medium, comprising a readable program for causing processing means
in or associated with a system comprising: a flow cell having an
inlet end and an outlet end, at least one sensing surface on a wall
surface within the flow cell located between the inlet and outlet
ends, wherein the flow cell has at least two inlet openings at the
inlet end and at least one outlet opening at the outlet end, such
that separate laminar flows entering the flow cell through the
respective inlet openings can flow side by side in the same
direction through the flow cell over the sensing surface, means for
applying laminar flows through the inlet openings, such that the
laminar flows pass side by side through the flow cell over the
sensing surface with an interface to each other that is parallel to
the direction of the laminar flows; and means for varying the
relative flow rates of the laminar fluids to displace laterally the
interface over the sensing surface; to control the execution of the
steps claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/384,626 filed May 31, 2002, and Swedish Patent
Application No. 0201637-6 filed May 31, 2002, both of which
applications are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method of coupling
binding agents to a substrate surface by passing a binding
agent-containing fluid flow over the surface, and more particularly
by using hydrodynamic addressing techniques to selectively direct
fluid flows to desired surface areas. The invention also relates to
analytical use of the method.
[0004] 2. Description of the Related Art
[0005] Flow cells are used extensively nowadays in a variety of
analytical systems. Typically, the flow cell has an inlet opening,
a flow channel with a sensing surface, and an outlet opening. A
sample fluid to be investigated is introduced through the inlet
opening, passes through the flow channel and leaves the flow cell
through the outlet opening. The flow cell may have more than one
inlet opening and optionally more than one outlet opening to permit
desired manipulations of the flow pattern within the flow cell.
[0006] The sensing surface usually comprises a substance layer to
which a recognition element for an analyte in the sample is
immobilised, typically a biochemical affinity partner to the
analyte. When the analyte interacts with the recognition element, a
physical or chemical change is produced on the sensing surface that
can be detected by a detector, e.g., an optical, electrochemical or
calorimetric detector. A flow channel may contain two or more
sensing surfaces with different recognition elements.
[0007] The sensing surface or surfaces in the flow cell may be
functionalized, or sensitized, in situ, i.e., within the flow cell.
WO 90/05305 discloses a method for functionalising a sensing
surface having functional groups thereon by passing a reagent
solution containing a bi- or polyfunctional ligand over the
surface, the ligand having a function which immobilizes the ligand
on the sensing surface and at least one more function which is
exposed on the sensing surface for interaction with the analyte. In
a specific embodiment, the sensing surface has a bound
carboxymethyl-dextran layer. After activation of the surface
through derivatization with N-hydroxysuccinimide, mediated by
N-ethyl-N'-(dimethylaminopropyl)carbodiimide, a ligand in the form
of aminotheophylline or aminobiotin is coupled to the activated
surface.
[0008] WO 99/36766 discloses methods and systems using hydrodynamic
addressing techniques to allow immobilization of different ligands
to discrete sensing areas within a single flow cell channel, as
well as to permit controlled sample delivery to such sensitized
areas. For a Y-type flow cell, which has an inlet end with two
inlet ports and an outlet end with one outlet port, and a sensing
surface between the ends, WO 99/36766 describes sensitization of
two spaced apart sensing areas with different ligands. This is done
by providing a laminar flow of a sensitising fluid adjacent to a
laminar flow of non-sensitizing (blocking) fluid such that the
fluids flow together over the sensing surface with an interface to
each other. By adjustment of the relative flow rates of the two
fluids the interface may be positioned laterally such that
sensitising fluid selectively contacts a desired area of the
sensing surface. More specifically, if the sensitizing fluid
initially contains a first ligand capable of binding to the sensing
surface and the interface is positioned such that the sensitizing
fluid covers, say, about one third of the lateral extension of the
sensing surface, and the blocking fluid covers the remaining two
thirds, the first ligand will be immobilized to the first-mentioned
third of the sensing surface. Then, the sensitizing fluid is
replaced by blocking fluid, and the blocking fluid is replaced by a
sensitizing fluid containing a second ligand. By positioning the
interface such that the sensitizing fluid again covers about one
third of the lateral extension of the sensing surface, now,
however, at the opposite side of the flow path, the second ligand
will be immobilized to that area, thereby providing a sensing
surface which, as seen laterally, has about one third immobilized
with the first ligand, about one third immobilized with the second
ligand, and an intermediate non-immobilized third which only has
been in contact with blocking fluid and may suitably be used as a
reference area.
[0009] For providing more than two differently sensitized sensing
surface areas, WO 99/36766 discloses the use of a so-called in-type
flow cell having three inlets and a single outlet. In this
embodiment, a laminar flow of sensitizing fluid is sandwiched
between two laminar flows of blocking fluid, and the sensitizing
fluid may thereby be displaced laterally to selectively contact a
number of sensing surface areas.
[0010] A similar use of a .psi.-type flow cell is disclosed in WO
00/56444.
[0011] While the .psi.-type flow cell is advantageous in comparison
with the Y-type flow cell in that the former readily permits
sensitization with more than two different ligands, the .psi.-type
flow cell requires the use of an additional pump (one pump for each
fluid flow inlet), which considerably complicates the control of
the different laminar flows. It would therefore be desired to be
able to use a Y-type flow cell to sensitize a sensing surface with
more than two different ligands.
[0012] Accordingly, it is an object of the present invention to
provide a method which permits coupling of multiple different
ligands, or generally binding agents, to respective surface areas
by hydrodynamic addressing using only two adjacent laminar fluid
flows, such as e.g., in a Y-type flow cell.
BRIEF SUMMARY OF THE INVENTION
[0013] The above and other objects and advantages are obtained by a
novel method of coupling multiple binding agents to respective
areas of a substrate surface by hydrodynamic addressing, wherein
each successive coupling of a binding agent to a substrate area is
followed or preceded by selective deactivation or activation of a
selected surface area or areas according to a particular coupling
protocol.
[0014] In one aspect, the present invention provides a method of
coupling at least two different binding agents to respective
defined areas of a substrate surface by hydrodynamic addressing
based on two laminar fluid flows that flow together in the same
direction over the substrate surface with an interface to each
other, and selectively contacting a defined area of the substrate
surface with a desired fluid by positioning the interface laterally
through adjustment of the relative flow rates of the two fluid
flows, which method comprises at least one of hydrodynamic
addressing procedures A and B, wherein procedure A comprises
immobilizing a first binding agent to a first area of the substrate
surface by contacting the area with a fluid containing the first
binding agent, deactivating the first area by subjecting the area
to a deactivating fluid, and immobilizing a second binding agent to
a second area of the substrate surface by contacting a substrate
surface area including the first and second areas with the second
binding agent; and procedure B comprises deactivating a first area
of the substrate surface by subjecting the area to a deactivating
fluid, immobilizing a first binding agent to a second area of the
substrate surface by contacting the first and second areas with a
fluid containing the first binding agent, activating at least a
part of the first area by subjecting the area to an activating
fluid, and immobilizing a second binding agent to the first area by
contacting the first area with a fluid containing the second
binding agent.
[0015] In one embodiment, the method comprises the steps of:
[0016] a) providing a substrate surface, at least a part of which
is reactive (e.g., activated) to permit coupling of binding agents
thereto;
[0017] b) passing over the substrate surface a laminar flow of a
fluid containing a first binding agent, and adjacent thereto a
laminar flow of a blocking fluid that does not interact with the
substrate surface such that the two fluids flow together in the
same direction with an interface to each other, and adjusting the
relative flow rates of the two laminar fluid flows to position the
interface such that the first binding agent-containing fluid
selectively contacts a first reactive (e.g., activated) area of the
substrate surface to couple the first binding agent thereto;
[0018] c) replacing the flow of the first binding agent-containing
fluid with a laminar flow of a deactivating fluid, and adjusting
the relative flow rates of the two laminar fluid flows to position
or displace the interface laterally such that the deactivating
fluid selectively contacts at least the first binding agent-coupled
area but less than the whole activated surface area for
deactivation thereof;
[0019] d) replacing the flow of the deactivating fluid with a
laminar flow of a fluid containing a second binding agent, and
adjusting the relative flow rates of the two laminar fluid flows to
displace the interface laterally such that the second binding
agent-containing fluid selectively contacts the deactivated area
and an adjacent second reactive (e.g., activated) area of the
substrate surface to selectively couple the second binding agent to
the second area; and, optionally,
[0020] e) replacing the flow of the second binding agent-containing
fluid with a laminar flow of deactivating fluid, and adjusting the
relative flow rates of the two laminar flows to position or
displace the interface laterally such that the deactivating fluid
selectively contacts at least the deactivated area and the second
binding agent-coupled area of the substrate surface for
deactivation thereof.
[0021] Another embodiment of the method comprises the steps of:
[0022] a) providing a substrate surface, at least part of which is
reactive to permit coupling of binding agents thereto;
[0023] b) passing over the substrate surface a laminar flow of a
deactivating fluid, and adjacent thereto a laminar flow of a
blocking fluid that does not interact with the substrate surface,
such that the two fluids flow together in the same direction with
an interface to each other, and adjusting the relative flow rates
of the two laminar fluid flows to position the interface such that
the deactivating fluid selectively contacts a first reactive area
of the substrate surface for deactivation thereof;
[0024] c) replacing the flow of the deactivating fluid with a
laminar flow of a fluid containing a first binding agent, and
adjusting the relative flow rates of the two laminar fluid flows to
displace the interface laterally such that the first binding
agent-containing fluid selectively contacts the deactivated area
and an adjacent second reactive area of the substrate surface to
selectively couple the first binding agent to the second area;
[0025] d) replacing the flow of the first binding agent-containing
fluid with a laminar flow of activating fluid, and adjusting the
relative flow rates of the two laminar fluid flows to displace the
interface laterally such that the activating fluid selectively
contacts at least a part of the deactivated first area for
activation thereof; and
[0026] e) replacing the flow of the activating fluid with a laminar
flow of a fluid containing a second binding agent, and adjusting
the relative flow rates of the two laminar fluid flows to position
or displace the interface laterally such that the second binding
agent-containing fluid selectively contacts the activated first
area to selectively couple the second binding agent thereto.
[0027] Optionally, combinations of the above two method embodiments
may also be used.
[0028] In another aspect, the present invention provides the use of
the method for analysing a fluid sample for the presence of at
least one analyte.
[0029] In still another aspect, the present invention provides the
use of the method for studying interactions of at least one analyte
with the substrate surface.
[0030] In yet another aspect, the present invention provides a
computer program product comprising program code means stored on a
computer readable medium or carried on an electrical or optical
signal for performing the method.
[0031] These and other aspects of this invention will be evident
upon reference to the attached drawings and the following detailed
description.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 is a schematic illustration of an embodiment of the
method of the present invention using a Y-type flow cell.
[0033] FIG. 2 is a schematic illustration of the different steps in
the method relating to FIG. 1.
[0034] FIG. 3 is a schematic illustration of another method
embodiment of the present invention using a Y-type flow cell.
DETAILED DESCRIPTION OF THE INVENTION
[0035] As mentioned above, the present invention is generally
directed to the coupling of multiple binding agents to a substrate
(solid support) surface using hydrodynamic addressing techniques to
successively bring binding agent-containing fluid flows into
selective contact with different surface areas to couple the
different binding agents thereto. More particularly, two adjacent
laminar fluid flows are provided which flow together over the
substrate surface with an interface between them, as described in
the aforementioned WO 99/36766 (the entire disclosure of which is
incorporated by reference herein).
[0036] By adjusting the relative flow rates of the two fluid flows,
the interface may be positioned laterally to make the different
fluids contact respective desired areas of the substrate surface.
To couple a binding agent to the substrate surface, one of the
fluid flows contains a binding agent, and the other fluid flow is
one that can not interact with the substrate surface, below
frequently referred to as a blocking fluid. To permit coupling of
the binding agent to the substrate surface, the surface should, of
course, be sufficiently reactive towards the binding agent.
Preferably, the surface is activated by an activating agent as is
per se well known in the art.
[0037] According to the present invention, a binding agent may be
coupled to a (preferably activated) surface followed by selective
deactivation of the coupled surface area, and optionally of an
adjacent area but less than the whole activated surface, and a
different binding agent is then coupled to an adjacent activated
surface area, followed by deactivation of the activated surface
area. By proceeding in this manner with successive coupling of
binding agents with deactivation after each coupling, multiple
binding agents may readily be coupled to respective areas of an
activated substrate surface, if desired with non-coupled areas
between the coupled areas.
[0038] Optionally, the procedure may start with deactivating an
edge area of the activated substrate surface, and a binding agent
is then coupled to an activated area adjacent to the deactivated
edge area.
[0039] While it is possible to carry out the coupling procedure of
the invention from one side of the flow path to the other, it may
be convenient to start from one side and successively couple a
first number of binding agents, and then shift to the opposite side
of the flow path and successively couple a second number of binding
agents from that side. In this case, activation of the surface area
to be coupled with this second number of agents may take place
after coupling the first number of binding agents.
[0040] Alternatively, a binding agent may be coupled to a
(preferably activated) surface by selective deactivation of a part
of the activated substrate surface, coupling of a binding agent to
the remaining activated area, reactivation of at least a part of
the deactivated area, and coupling of a different binding agent to
the reactivated area. By proceeding in this way with successive
activation and deactivation before each coupling of binding agent,
multiple binding agents may be immobilized to respective areas of
an activated surface.
[0041] Optionally, a pre-activated surface may be used instead of
activating the surface areas at the time of coupling the binding
agents.
[0042] It is not necessary that all the binding agents coupled to
the substrate surface be different. However, usually at least two
adjacent binding agent coupled areas should not support the same
binding agent.
[0043] Preferably, at least one surface area is not coupled with
binding agent and used as a reference area(s).
[0044] The term "binding agent" as used herein means any agent that
is a member of a specific binding pair, including, for instance
polypeptides, such as, e.g., proteins or fragments thereof,
including antibodies; nucleic acids, e.g., oligonucleotides,
polynucleotides, and the like; etc. The binding agent is often a
ligand.
[0045] The term "ligand" as used herein means a molecule that has a
known or unknown affinity for a given analyte and can be
immobilized on a predefined region of the surface. The ligand may
be a naturally occurring molecule or one that has been synthesized.
The ligand may be used per se or as aggregates with another
species. Optionally, the ligand may also be a cell.
[0046] The term "reactive" with respect to a substrate surface
means that the surface should exhibit a binding moiety, such as
e.g., a functional group, capable of coupling to a binding
agent.
[0047] The term "activation" means modification of a substrate
surface to enable coupling a binding agent thereto, usually
modification of a functional group on the substrate surface.
[0048] The term "deactivation" means modification of a reactive
substrate surface, usually of an activated functional group
thereon, such that coupling of a binding agent to the surface is
substantially prevented. Deactivation may include restoring an
original functional group or making a reactive functional group or
other reactive moiety inactive in other ways.
[0049] Activating and deactivating agents that may be used for the
purposes of the present invention are per se well known to a person
skilled in the art and may readily be selected for each particular
situation.
[0050] The choice of activating agent (and method) depends, of
course, on the functional group to be activated and on the desired
reactive group to be obtained by the activation, which in turn
depends on the binding agent to be coupled to the substrate
surface. Exemplary functional group/activating agent combinations
include those introducing hydroxysuccinimide esters, nitro- and
dinitrophenyl esters, tosylates, mesylates, triflates and
disulfides. For example, a hydroxy group may be reacted to
activated ester with disuccinic carbonate, or to epoxide with a
diepoxide. A carboxy group may be activated to N-hydroxysuccinimide
ester by reaction with N-hydroxysuccinimide (NHS) and carbodiimide,
e.g., 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide (EDC), or to
dinitrophenyl ester by reaction with dinitrophenol. A thiol
(mercapto) group may be activated to a disulfide group by reaction
with e.g., a dipyridyldisulfide or
(2-pyridinyldithio)ethaneamine.
[0051] For example, NHS/EDC activation of carboxy groups may be
used to couple binding agents having an amine function (so-called
amine coupling) or an aldehyde function (so-called aldehyde
coupling).
[0052] NHS/EDC activation may also be used to introduce thiol
groups, e.g., by reaction with dithioerythritol (DTE). These thiol
groups may then either (i) be reacted with an active disulfide
group of a binding agent (so-called surface thiol coupling), or
(ii) be activated to disulfide groups which may be reacted with a
thiol function of a binding agent (so-called ligand thiol
coupling).
[0053] Also avidin or streptavidin may be coupled to an
NHS/EDC-activated surface to permit capture of a biotinylated
binding agent (so-called avidin coupling).
[0054] The choice of deactivating agent depends, of course, on the
active group(s) to be deactivated. For example,
N-hydroxysuccinimide esters may be deactivated with ethanolamine or
sodium hydroxide, deactivation with sodium hydroxide being
reversible, i.e., the deactivated surface may be reactivated by
activation with an activating agent.
[0055] The term "coupling" as used herein is to be interpreted in a
broad sense and includes covalent binding as well as other types of
binding.
[0056] The method of the invention is preferably performed in a
flow cell. Suitable flow cells for use in the present invention may
assume a number of forms, the design of which may vary widely. A
preferred type of flow cell is the "Y-flow cell" which has two
inlets and one outlet and which is described in, for example, the
above-mentioned WO 99/36766.
[0057] The substrate surface is usually a sensing surface, which
term in the present context is to be construed broadly. The sensing
surface may, for example, be a surface or surface layer that can
interact specifically with a species present in a fluid, a surface
or surface layer that can be chemically or physically sensitised to
permit such interaction, or a surface or surface layer that can be
chemically or physically activated to permit sensitisation thereof.
A flow cell may contain one or more sensing surfaces.
[0058] Binding events at the sensing surface may be detected by
numerous techniques. In many cases it is favourable to use
so-called non-label methods. Representative such detection methods
include, but are not limited to, mass detection methods, such as
piezoelectric, optical, thermo-optical and surface acoustic wave
(SAW) device methods, and electrochemical methods, such as
potentiometric, conductometric, amperometric and
capacitance/impedance methods. With regard to optical detection
methods, representative methods include those that detect mass
surface concentration, such as reflection-optical methods,
including both external and internal reflection methods, angle,
wavelength, polarization, or phase resolved, for example evanescent
wave ellipsometry and evanescent wave spectroscopy (EWS, or
internal reflection spectroscopy), both may include evanescent
field enhancement via surface plasmon resonance (SPR), Brewster
angle refractometry, critical angle refractometry, frustrated total
reflection (FTR), scattered total internal reflection (STIR), which
may include scatter enhancing labels, optical wave guide sensors;
external reflection imaging, evanescent wave-based imaging such as
critical angle resolved imaging, Brewster angle resolved imaging,
SPR-angle resolved imaging, and the like. Further, photometric and
imaging/microscopy methods, "per se" or combined with reflection
methods, based on for example surface enhanced Raman spectroscopy
(SERS), surface enhanced resonance Raman spectroscopy (SERRS),
evanescent wave fluorescence (TIRF) and phosphorescence may be
mentioned, as well as waveguide interferometers, waveguide leaking
mode spectroscopy, reflective interference spectroscopy (RIfS),
transmission interferometry, holographic spectroscopy, and atomic
force microscopy (AFR).
[0059] SPR spectroscopy may be mentioned as an exemplary
commercially available analytical system to which the present
invention may be applied. One type of SPR-based biosensors is sold
by Biacore AB (Uppsala, Sweden) under the trade name BIACORE.RTM..
These biosensors utilize a SPR based mass-sensing technique to
provide a "real-time" binding interaction analysis between a
surface bound ligand and an analyte of interest.
[0060] The basic principles of the invention will now be further
described with reference to FIG. 1. A Y-type flow cell, generally
designated by reference numeral 1, has two inlets 2 and 3,
respectively, and an outlet 4. The flow cell has a sensing surface
5 on a wall portion thereof. The sensing surface may, for example,
be of the type described in U.S. Pat. Nos. 5,242,828 and 5,436,161
(the full disclosures of which are incorporated by reference
herein) and may, for instance, include a matrix coating in the form
of carboxymethylated dextran.
[0061] A laminar flow of a first fluid, indicated by arrow 6, is
introduced through inlet 2, and a laminar flow of a second fluid,
indicated by arrow 7, is introduced through inlet 3 such that the
two fluids flow together over the sensing surface 5 with an
interface (not shown) between them, exiting through outlet 4, as
indicated by arrow 8. By adjusting the relative flow rates of the
two fluid flows, the position of the interface may be displaced
laterally as desired and be set at any distance from either side
wall of the flow cell. Immobilization of three different ligands to
the sensing surface 5 is performed as described in steps (1) to
(10) below. Reference is simultaneously made to FIG. 2, which
schematically illustrates the procedure. Each square in FIG. 2
represents a Y-cell, as shown in FIG. 1, with the respective
process step number indicated at the top thereof.
[0062] (1) The procedure is started with the first fluid 6 being a
fluid containing an activating agent, and the second fluid 7 being
a blocking fluid (i.e., one that does not affect or interact with
the sensing surface), such as buffer. The interface between the two
fluid flows is positioned such that the activating fluid
selectively covers a sensing surface area which, in the illustrated
case, extends over more than half the lateral extension of the
sensing surface as indicated by arrow 9. If the sensing surface 5
includes a layer of carboxymethylated dextran as suggested above,
the activating agent may be N-hydroxysuccinimide (NHS) together
with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC). It is,
alternatively, possible to activate the whole sensing surface by
replacing the two fluid flows by a single flow of activating
fluid.
[0063] (2) The activating fluid is then replaced by a fluid
containing a deactivating agent, and the interface between the two
fluid flows 6, 7 is positioned at the point of arrow 10, such that
the deactivating fluid selectively covers an area 11 close to the
flow cell wall 12, the buffer fluid covering the rest of the
sensing surface 5. The area 11 will thereby be deactivated so as
not to react with ligand-containing fluid in the following step. If
NHS/EDC is used as activating agent, the deactivating agent may,
for example, be ethanolamine.
[0064] (3) The deactivating fluid 6 is then replaced by a fluid
containing a first ligand, e.g., a first monoclonal antibody, and
the interface between the two fluid flows 6, 7 is positioned at the
point of arrow 13, such that the ligand-containing fluid
selectively covers the deactivated area 11 as well as an adjacent
activated area 14. The area 14 will thereby have the first ligand
coupled thereto.
[0065] (4) The ligand-containing fluid 6 is then replaced by
deactivating fluid, and the interface between the two fluid flows
6, 7 is positioned at the point of arrow 15, such that the
deactivating fluid selectively covers areas 11, 14 and an adjacent
activated area 16 which will thereby be deactivated.
[0066] (5) The deactivating fluid 6 is then replaced by a fluid
containing a second ligand, e.g., a second monoclonal antibody, and
the interface between the two fluid flows 6, 7 is positioned at the
point of arrow 9, such that the ligand-containing fluid covers
areas 11, 14, 16 and an adjacent activated area 17 extending
between the points of arrows 9 and 15. The area 17 will thereby
have the second ligand coupled thereto.
[0067] (6) The ligand-containing fluid 6 is then replaced by a
deactivating fluid, and the interface between the two fluid flows
6, 7 is positioned at the point of arrow 9, such that the
deactivating fluid covers areas 11, 14, 16 and 17 which will
thereby be deactivated.
[0068] (7) The ligand-containing fluid 6, introduced through inlet
2, is then replaced by buffer, and the buffer flow 7, introduced
through inlet 3, is replaced by activating fluid. The interface
between the two fluid flows 6, 7 is positioned such that the
activating fluid selectively covers an area extending as indicated
by arrow 18, i.e., from the flow cell wall 19 towards but not up to
the ligand-coupled area 17.
[0069] (8) The activating fluid 7 is then replaced by deactivating
fluid, and the interface between the two fluid flows 6, 7 is
positioned at the point of arrow 20 such that the deactivating
fluid selectively covers an activated area 21 close to the flow
cell wall 19 which area is thereby deactivated.
[0070] (9) The deactivating fluid 7 is then replaced by a fluid
containing a third ligand, e.g., a third monoclonal antibody, and
the interface between the two fluid flows 6, 7 is positioned at the
point of arrow 19, such that the ligand-containing fluid covers the
area 21 and an adjacent activated area 22 extending between the
points of arrows 18 and 20. This area 22 will thereby have the
third ligand coupled thereto.
[0071] (10) Finally, the ligand-containing fluid 7 is replaced by
deactivating fluid, and the interface between the two fluid flows
6, 7 is positioned at the point of arrow 9 such that the
deactivating fluid 7 selectively covers the deactivated area 21,
the ligand-coupled area 22 and (in the illustrated case) a
non-activated area 23 between the ligand-coupled areas 22 and 17
for deactivation thereof. (Alternatively, the deactivating fluid
may only cover the areas 21 and 22).
[0072] The sensing surface 5 now exhibits three discrete sensing
areas 14, 17 and 22, each supporting a different ligand, which
sensing areas are separated mutually as well as to the flow cell
walls 12, 19 by deactivated areas 11, 16, 21 and 23. The resulting
Y-cell with the desired sensitized sensing surface is also shown in
FIG. 2, to the right of the step 10 Y-cell.
[0073] While in the above illustrated case three discrete areas
have been immobilised with ligand, it is understood that by
proceeding as described above with successive ligand-coupling and
deactivation steps, from e.g., four, five or six to considerably
more ligand-coupled areas may likewise readily be produced on a
sensing surface, depending on among other things the flow cell, the
size of the sensing surface, the precision and control of pumps,
etc.
[0074] In the above described immobilisation procedure, the
different sensing surface areas are immobilised from the edges of
the surface towards the centre. This requires, however, that
ligands have to be contacted with areas to which a ligand has
already been immobilised. This may be avoided by a procedure in
which the ligands are immobilised to the respective areas from the
centre towards the edges. An embodiment of such a procedure is
outlined below with reference to FIG. 3.
[0075] In FIG. 3, the flow cell 30 is a Y-cell similar to that
shown in FIGS. 1 and 2, having two inlets 31, 32, an outlet 33 and
a sensing surface on a wall portion thereof (not specifically
indicated). Three detection spots, i.e., areas on the sensing
surface that are measured by a detection system (not shown) are
indicated at 34, 35 and 36. A number of images of flow cell 30,
numbered from 1 to 9, are shown to illustrate the different steps
of the procedure. The sensing surface may e.g., be
carboxymethylated dextran, the activating fluid may be an
NHD/EDC-solution, deactivating fluids may be sodium hydroxide
solution and ethanolamine solution, and the blocking fluid
buffer.
[0076] (1) The procedure is started by passing a laminar flow of
activating fluid through inlet 31 and blocking fluid through inlet
32. In the illustrated case, the interface between the two fluid
flows is positioned such that the activating fluid covers detection
spots 34 and 35 (i.e., two thirds of the lateral extension of the
sensing surface).
[0077] (2) The activating fluid is then replaced by deactivating
fluid, and the interface is positioned such that the deactivating
fluid covers detection spot 34 (i.e., the left third of the sensing
surface) to deactivate that part of the activated surface area,
leaving the centre area that includes detection spot 35 activated.
The deactivating fluid is selected such that the deactivated area
may later be reactivated (e.g., sodium hydroxide as mentioned
above).
[0078] (3) The deactivating fluid is then replaced by a fluid
containing a first ligand, and the interface is positioned such
that the ligand-containing fluid covers detection spots 34 and 35,
i.e., the same area as that activated in step (1) above. Due to the
deactivating step (2), the first ligand will only couple to the
still activated centre area including detection spot 35.
[0079] (4) The ligand-containing fluid is then replaced by
activating fluid, and the interface is positioned such that the
activating fluid covers the area that was deactivated in step (2)
above (i.e., the left third of the sensing surface in FIG. 3).
[0080] (5) The activating fluid is then replaced by a fluid
containing a second ligand, and the interface is positioned such
that ligand-containing fluid covers the area that was activated in
step (4) above. Thereby, the second ligand will be coupled to this
area. The sensing surface now has the second ligand coupled to the
left area that includes detection spot 34, and the first ligand to
the centre area that includes detection spot 35.
[0081] (6) The ligand-containing fluid supplied through inlet 31 is
then replaced by blocking fluid, and the blocking fluid supplied
through inlet 32 is replaced by activating fluid. The interface is
now positioned such that the activating fluid covers the right
third of the sensing surface to activate that area.
[0082] (7) The activating fluid is then replaced by a fluid
containing a third ligand, and the interface is maintained in the
same position as in step (6) above to thereby couple the third
ligand to the area that was activated in step (4) and includes
detection spot 36.
[0083] (8) The activating fluid through inlet 31 is then replaced
by deactivating fluid and inlet 32 is closed to deactivate all
three ligand-coupled areas of the sensing surface. In this case the
deactivating fluid need not be one that permits reactivation as in
step (2) and must not affect the immobilised ligands, e.g.,
ethanolamine.
[0084] (9) The sensing surface now has three different ligands
coupled thereto, i.e., the second ligand coupled to a lateral area
including detection spot 34, the first ligand coupled a central
area including detection spot 35, and the third ligand coupled to a
lateral area including detection spot 36.
[0085] It is readily seen that ligands may readily be coupled to
more than three different areas by following the above-described
procedure.
[0086] An alternative immobilization procedure, which partly avoids
contacting a ligand with an area or areas to which ligands have
already been immobilized, and does not require reactivation of a
deactivated surface, will be schematically described below with
reference to FIG. 3 again.
[0087] (1) Half the sensing surface is first activated by
introducing activating fluid through inlet 31 and blocking fluid
through inlet 32.
[0088] (2) A first ligand is then immobilized to an inner part
(i.e., adjacent to the centre) of the activated area by introducing
ligand through inlet 32 and blocking fluid through inlet 31, and
positioning the fluid interface in the middle of the activated
area.
[0089] (3) A second ligand is then immobilized to the remaining
part of the activated area (i.e., adjacent to the flow cell wall)
by introducing ligand through inlet 31 and blocking fluid through
inlet 32.
[0090] (4) After deactivation of the areas coupled with the first
and second ligands, respectively, third and fourth ligands may then
be immobilized to the other half of the sensing surface by
activating this area and successively coupling the ligands,
introduced through inlet 31, according to the method variant
described above with reference to FIGS. 1 and 2, i.e., with
intermediate deactivation of the inner area coupled with the third
ligand before introducing the fourth ligand.
[0091] Alternatively, a third ligand may be coupled to the
remaining part of the activated area by introducing the ligand
through inlet 32.
[0092] By proceeding as described above with reference to FIG. 3,
it is also possible to couple a fourth ligand without the
ligand-containing fluid having to pass areas with immobilized
ligands, i.e., by deactivating an outer part of the activated area
(adjacent to the flow cell wall) prior to the introduction of the
third ligand through inlet 32, reactivating the deactivated area
after coupling of the third ligand, and subsequently introducing
the fourth ligand through inlet 32 to couple the ligand to the
reactivated area.
[0093] Numerous applications of a sensing surface to which multiple
ligands have been immobilised at discrete areas as described above
are readily apparent to a person skilled in the art and need not be
detailed herein.
[0094] The invention will now be illustrated further by the
following non-limiting examples.
EXAMPLE 1
[0095] Immobilization of Three Different Ligands in a Y-Cell
[0096] A BIACORE.RTM. S51 instrument (Biacore AB, Uppsala, Sweden)
was used. This instrument has two Y-type flow cells which allow a
dual flow of fluids over a sensor chip surface, so-called
hydrodynamic addressing, as described in WO 99/36766 mentioned
above. The instrument uses three parallel detection spots on the
sensor chip, each detection spot occupying one diode row in a diode
array detector for detecting light reflected at the detection spots
on the sensor chip surface. The detection spots are mutually spaced
by one diode row. As sensor chip was used Sensor Chip CM5 (Biacore
AB, Uppsala, Sweden) which has a gold-coated surface with a
covalently linked carboxymethyl-modified dextran polymer hydrogel.
Running buffer was HBS-N (10 mM HEPES pH 7.4 and 150 mM NaCl)
(Biacore AB, Uppsala, Sweden). The output from the instrument is a
"sensorgram" which is a plot of detector response (measured in
"resonance units", RU) as a function of time. An increase of 1000
RU corresponds to an increase of mass on the sensor surface of
approximately 1 ng/mm.sup.2.
[0097] Antibodies anti-IL-8, anti-IL-10 and anti-IL-12 against
interleukin 8 (IL-8), interleukin 10 (IL-10) and interleukin 12
(IL-12) (in-house sources, Biacore AB, Uppsala, Sweden), were
diluted 10 times from stock solution with 10 mM acetate pH 4.5.
They were then immobilized in the two flow cells of the instrument
by the hydrodynamic addressing procedure described above with
reference to FIGS. 1 and 2, using 10 minutes activation with
EDC/NHS and sequential deactivation and ligand (antibody)
immobilization. Each deactivation was performed with ethanolamine
(100 mM, pH 8.5) for two minutes, and each antibody was injected
for 7 minutes.
[0098] The different antibodies were immobilized in parallel
stripes extending through the spots used for detection in the
BIACORE.RTM. S51 instrument, meaning that with regard to the
detector there was one diode row between each immobilized spot.
However, in order to ensure that the immobilized antibodies within
the detection spots were homogenous, each antibody was immobilized
such that it extended into one third of the adjacent interspaces.
The resulting immobilization levels in resonance units (RU) for the
different ligands are shown in Tables 1 (flow cell 1) and 2 (flow
cell 2) below. Bold values show targeted immobilization spots.
Indicated in italics below each immobilization level value is the
percentage immobilized, obtained by subtracting the baseline before
injection of antibody from the baseline after subsequent
deactivation with ethanolamine.
1TABLE 1 Immobilized Diode row number (flow cell 1) antibody 1 2 3
4 5 anti-IL-8 31480 18821 280 189 116 100.0 46.0 -0.3 -0.5 -0.1
anti-IL-10 31917 28574 26629 18645 62 -14.4 32.2 100.0 47.7 -0.1
anti-IL-12 23892 14945 13037 9904 15611 -6.1 -20.3 -13.2 21.2
100.0
[0099]
2TABLE 2 Immobilized Diode row number (flow cell 2) antibody 1 2 3
4 5 anti-IL-8 123 152 147 20214 30138 -0.1 -0.3 -0.2 49.6 100.0
anti-IL-10 98 17142 26016 27174 30496 0.1 49.3 100.0 20.6 -13.0
anti-IL-12 16013 9407 13525 13877 23681 100.0 14.5 -8.7 -18.8
-12.0
[0100] As shown in the tables, selective repeatable immobilization
of ligands at the desired detection spots was obtained with no or
very low "cross-talk" between spots.
[0101] The corresponding antigens IL-8, IL-10 and IL-12 were
diluted to 500 ng/ml in HBS-N (Biacore AB, Uppsala, Sweden) and
sequentially injected for 4 minutes in both flow cells. Between
injections, the surfaces were regenerated with 0.1 trifluoroacetic
acid (TFA) for 6 seconds. The resulting binding levels (in RU) are
shown in Tables 3 and 4 below. Below each binding value, calculated
percent cross-talk is indicated in italics.
3 TABLE 3 Analyte Diode row number (flow cell 1) antigen 1 2 3 4 5
IL-8 1611 776 25 14 2 100.0 48.2 1.5 0.9 0.1 IL-10 2 287 484 6 -1
0.5 59.4 100.0 1.3 -0.3 IL-12 1 4 8 450 1076 0.1 0.4 0.8 41.8
100.0
[0102]
4 TABLE 4 Analyte Diode row number (flow cell 2) antigen 1 2 3 4 5
IL-8 4 16 26 730 1451 0.3 1.1 1.8 50.3 100.0 IL-10 -3 8 328 272 1
-0.9 2.5 100.0 83.1 0.3 IL-12 1183 396 7 2 -2 100.0 33.4 0.6 0.2
-0.2
[0103] As shown in the tables above, cross-talk between spots was
below 1%, except for IL-8 which bound by 1.5% and 1.8%,
respectively, to the anti-IL-10 antibody, most likely due to
cross-reactivity.
EXAMPLE 2
[0104] Immobilization of Five Different Ligands in a Y-Cell
[0105] Following the procedure in Example 1 and using the same
instrument and sensor chip, five different ligands were immobilized
in a Y-cell. In this case, however, each ligand spot adjoined to
the next one without any separating detector diode row. The
interfaces between the spots were adjusted to obtain a 5%
theoretical cross-talk to ensure homogeneity of the spots. The
following ligands were sequentially immobilized: anti-IL-8,
anti-myoglobin, anti-IL-10, anti-CKMB and anti-IL-12 (all from
in-house sources, Biacore AB, Uppsala, Sweden). The resulting
immobilization levels for flow cell 1, as well as the percentages
cross-talk (in italics), are shown in Table 5 below.
5TABLE 5 Immobilized Diode row number (flow cell 2) antibody 1 2 3
4 5 anti-IL-8 27550 910 253 180 127 3 1 1 0 anti- 32199 29280 1149
182 138 myoglobin 3 0 0 anti-IL-10 29762 26385 24325 1024 123 4 0
anti-IL-12 22680 15940 12330 1763 24848 1 0 0 -2 anti-CKMB 22769
16003 12690 19940 26576 0 0 2
[0106] As appears from the table above, all the antibody
immobilizations were successful, the maximum cross-talk obtained
being 4%.
[0107] The antigens corresponding to the immobilized antibodies
were then injected as described in Example 1, except that myoglobin
and CKMB were each diluted to 5 .mu.g/ml in HBS-N. The resulting
binding level for each analyte in percent of that for the "active"
spot (after subtraction of the average for buffer samples) are
presented in Table 6 below.
6 TABLE 6 Analyte Diode row number (flow cell 2) antigen 1 2 3 4 5
myoglobin 2.9 100.0 9.3 0.5 0.0 CKMB -2.4 -2.5 0.5 100.0 3.7 IL-8
100.0 3.9 1.8 1.0 -0.1 IL-10 -2.6 -2.2 100.0 -0.3 -2.8 IL-12 -0.4
-0.4 1.5 3.1 100.0
[0108] As shown in the table above, the maximum cross-talk was
below 10%.
[0109] It is to be understood that the invention is not limited to
the particular embodiments of the invention described above, but
the scope of the invention will be established by the appended
claims.
* * * * *