U.S. patent application number 11/249038 was filed with the patent office on 2006-05-04 for preparation and use of a reactive solid support surface.
This patent application is currently assigned to Biacore AB. Invention is credited to Tanja Jarhede, Per Kjellin, Anita Larsson, Hans Sjobom.
Application Number | 20060094060 11/249038 |
Document ID | / |
Family ID | 33434247 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094060 |
Kind Code |
A1 |
Jarhede; Tanja ; et
al. |
May 4, 2006 |
Preparation and use of a reactive solid support surface
Abstract
A method of preparing a protein-resistant reactive solid support
surface is disclosed. The method comprises the steps of providing a
solid support having a hydrogel coating with a plurality of binding
elements, coupling a protein resistant compound to the hydrogel via
a first fraction of the binding elements, and coupling at least one
binding agent to the hydrogel via a second fraction of the binding
elements, whereby the protein resistant compound and the at least
one binding agent are co-immobilized to the hydrogel. Also the use
of the reactive surface in analysis, such as immunogenicity assays,
is disclosed.
Inventors: |
Jarhede; Tanja; (Storvreta,
SE) ; Kjellin; Per; (Uppsala, SE) ; Larsson;
Anita; (Uppsala, SE) ; Sjobom; Hans; (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
SE-754 50
|
Family ID: |
33434247 |
Appl. No.: |
11/249038 |
Filed: |
October 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60618655 |
Oct 13, 2004 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
427/2.11 |
Current CPC
Class: |
B82Y 30/00 20130101;
G01N 2800/52 20130101; Y10S 436/809 20130101; G01N 33/548
20130101 |
Class at
Publication: |
435/007.1 ;
427/002.11 |
International
Class: |
G01N 33/53 20060101
G01N033/53; B05D 3/00 20060101 B05D003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 2004 |
SE |
0402476-6 |
Claims
1. A method of preparing a reactive solid support surface, which
method comprises the steps of: a) providing a solid support having
a hydrogel coating with a plurality of binding elements; b)
coupling a protein resistant compound to the hydrogel via a first
fraction of the binding elements; and c) coupling at least one
binding agent to the hydrogel via a second fraction of the binding
elements; whereby the protein resistant compound and the at least
one binding agent are co-immobilized to the hydrogel.
2. The method according to claim 1, wherein step c) is performed
after step b).
3. The method according to claim 2, wherein the second fraction of
the binding elements are comprised by those remaining on the
hydrogel after coupling of the protein resistant compound via the
first fraction of the binding elements.
4. The method according to claim 1, wherein the binding elements of
the hydrogel comprise functional groups, and the protein resistant
compound and the at least one binding agent are covalently coupled
to the hydrogel via the functional groups.
5. The method according to claim 4, wherein the protein resistant
compound and the at least one binding agent are coupled to the same
kind of functional group of the hydrogel.
6. The method according to claim 4, wherein the functional groups
are selected from hydroxyl, carboxyl, amino, aldehyde, carbonyl,
epoxy, vinyl and thiol.
7. The method according to claim 4, wherein in at least one of
steps b) and c) of claim 1 functional groups are activated by an
activating agent prior to the coupling.
8. The method according to claim 7, wherein the activated
functional groups are selected from reactive ester, hydrazide,
thiol, maleimide and reactive disulphide-containing derivative.
9. The method according to claim 4, wherein the protein resistant
compound and the binding agent each contain a functional group
independently selected from amine, thiol and a reactive
disulphide-containing derivative.
10. The method according to claim 1, wherein the binding elements
of the hydrogel comprise one member of a specific binding pair and
the protein resistant compound contains the other member of the
specific binding pair.
11. The method according to claim 1, wherein the protein resistant
compound is a hydrophilic polymer.
12. The method according to claim 1, wherein the protein resistant
compound comprises poly(ethylene glycol) or a derivative
thereof.
13. The method according to claim 1, wherein the hydrogel is a
polysaccharide.
14. The method according to claim 13, wherein the polysaccharide is
selected from agarose, dextran, carrageenan, alginic acid, starch,
cellulose, and derivatives thereof.
15. The method according to claim 13, wherein the polysaccharide is
dextran or a derivative thereof.
16. The method according to claim 13, wherein the polysaccharide is
cellulose or a derivative thereof.
17. The method according to claim 1, wherein the binding agent is a
ligand capable of specifically binding to an analyte.
18. The method according to claim 1, wherein the binding agent is a
capture agent capable of binding to an analyte-specific ligand.
19. The method according to claim 1, wherein the amount of protein
resistant compound that is coupled in step b) of claim 1 is not
less than about 2 ng/mm.sup.2.
20. The method according to claim 1, wherein the amount of binding
agent that is coupled in step c) of claim 1 is not less than about
9 ng/mm.sup.2.
21. The method according to claim 1, wherein the solid support
surface to which the hydrogel is attached comprises a metal
layer.
22. The method according to claim 21, wherein the hydrogel is
attached to the metal layer via an ordered monolayer of alkyl
chains.
23. The method according to claim 1, wherein the hydrogel coating
on the solid support comprises an array of defined discrete
areas.
24. The method according to claim 1, wherein the solid support
surface comprises a sensor surface.
25. Use of a reactive solid support surface prepared according to
the method of claim 1 for analysis of at least one analyte in a
sample.
26. The use according to claim 25, wherein at least one of (i)
analyte concentration and (ii) a kinetic parameter for at least one
interaction of analyte with ligand is determined.
27. The use according to claim 25, wherein the sample is selected
from blood serum, blood plasma and whole blood.
28. The use according to claim 25, wherein the analyte is a
protein.
29. The use according to claim 25, wherein the analysis comprises
detection based on mass sensing.
30. The use according to claim 25, wherein the analysis comprises
an immunogenicity study.
31. A method of determining if an immune response against an
antigen has occurred in a mammal, which method comprises
determining the presence of antibodies against the antigen in a
blood sample by a surface sensitive detection technique using a
sensor surface with a hydrogel coating which has co-immobilized
thereon an antibody specific binding agent and a protein resistant
compound.
32. A protein resistant solid support surface for coupling of
binding agents, comprising a hydrogel layer containing a plurality
of binding elements, wherein a selected fraction of the binding
elements are coupled to a protein resistant compound, and the
remaining binding elements are free for coupling of at least one
binding agent to the solid support.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 60/618,655 filed
Oct. 13, 2004; and also claims priority to Swedish Application No.
0402476-6 filed Oct. 13, 2004; both of these 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 for preparing a
solid support surface having binding agents, such as
analyte-specific ligands, immobilized thereto, and more
particularly to such a surface which resists non-specific binding.
The invention also relates to the use of the prepared solid support
surface in analysis, such as immunogenicity assays, and to a
protein-resistant solid support surface for coupling of binding
agents thereto.
[0004] 2. Description of the Related Art
[0005] A variety of analytical techniques are used to characterize
interactions between molecules, particularly in the context of
assays directed to the detection and interaction of biomolecules.
For example, antibody-antigen interactions are of fundamental
importance in many fields, including biology, immunology and
pharmacology. In this context, many analytical techniques involve
binding of a "ligand", such as an antibody, to a solid support,
followed by contacting the ligand with an "analyte", such as an
antigen. Following contact of the ligand and analyte, some
characteristic is measured which is indicative of the interaction,
such as the ability of the ligand to bind the analyte. It is often
desired that after measurement of the interaction, it should be
possible to dissociate the ligand-analyte pair in order to
"regenerate" free ligand, thereby enabling reuse of the ligand
surface for a further analytical measurement.
[0006] Analytical sensor systems that can monitor such molecular
interactions in real time are gaining increasing interest. These
systems are often based on optical biosensors and usually referred
to as interaction analysis sensors or biospecific interaction
analysis sensors. A representative such biosensor system is the
Biacore.RTM. instrumentation sold by Biacore AB (Uppsala, Sweden),
which uses surface plasmon resonance (SPR) for detecting
interactions between molecules in a sample and molecular structures
immobilized on a sensing surface. With the Biacore.RTM. systems it
is possible to determine in real time without the use of labeling
not only the presence and concentration of a particular molecule in
a sample, but also additional interaction parameters such as, for
instance, the association rate and dissociation rate constants for
the molecular interaction.
[0007] Since the SPR-based detection, like several other so-called
label-free detection techniques, senses mass changes at the sensor
surface, non-specific binding to the sensor surface will also be
sensed, giving a false addition to the detected response at the
surface. This is particularly the case where the samples are
complex mixtures such as a blood serum or crude cell extract. The
non-specific binding may arise from binding of non-analyte
molecules in the sample to the immobilized ligand or from
non-specific binding of analyte or non-analyte molecules to the
actual sensor surface. In, for example, immunogenicity studies
where antibodies in serum samples are analyzed, problems in most
cases arise due to non-specific binding from non-analyte species in
the serum, this non-specific binding often being greater than the
specific binding of the target antibody.
[0008] It is known that poly(ethylene glycol) (PEG) coatings may
significantly reduce the non-specific adsorption of proteins and
cells to a surface.
[0009] U.S. Pat. No. 6,475,808 discloses an assay device comprising
a substrate with a surface having an array of discrete
array-regions. An ordered hydrophobic monolayer of alkyl chains is
chemisorbed or physisorbed to the surface, and a hydrophilic
monolayer formed from poly(ethylene glycol) chains is covalently
linked to the hydrophobic monolayer. A plurality of
protein-immobilizing groups are covalently attached to a selected
fraction of the poly(ethylene glycol) chains within the array
regions. The hydrophobic monolayer and the poly(ethylene glycol)
chains are effective in combination to resist non-specific protein
binding.
[0010] WO 2004/005477 discloses a microarray which comprises a
substrate having a substantially planar surface comprising an
organic-chemically modified dielectric-coated reflective metal,
e.g., gold, and a plurality of proteins stably attached to the
surface via a chemical adapter, e.g., a functionalized dextran.
After spotting the proteins onto the substrate surface, the surface
may be derivatized with poly(ethylene glycol) or a poly(ethylene
glycol) analogue to inhibit non-specific protein adsorption.
[0011] WO 03/005890 discloses an optical fiber surface plasmon
resonance (SPR) sensor having a gold surface to which a dextran
layer is bound via a self-assembled monolayer (SAM) of
11-mercapto-dodecanol. Anti-myoglobin antibodies are attached to
the dextran via carboxylated hydroxyl groups thereof. To eliminate
non-specific binding to the sensor, thiol-terminated poly(ethylene
glycol), e.g., methoxy-PEG-thiol, may be coupled to the gold
surface through a gold-thiol bond or to the dextran. Immobilized
PEG surrounding the sensor will prevent non-specific interactions
with the surface while allowing specific receptor-ligand
interactions.
[0012] The present invention seeks to provide an improved method
for preparing a protein-resistant solid support surface having
binding agents immobilized thereon.
BRIEF SUMMARY OF THE INVENTION
[0013] The above and other objects and advantages are provided by a
method for preparing a solid support surface with immobilized
binding agent(s) and a protein resistant compound, which method
basically is characterized in that the solid support surface has a
hydrogel coating, and that (i) a protein resistant compound, such
as, e.g., poly(ethylene glycol) or a derivative thereof, and (ii) a
binding agent(s) are coupled to the hydrogel to be co-immobilized
thereon. In this way, it is possible to provide a solid support
surface, such as a sensing surface, which has a high level of
binding agent, e.g., analyte-specific ligand, simultaneously with a
sufficient level of a protein resistant compound to effectively
resist non-specific binding.
[0014] In one aspect, the present invention therefore provides a
method of preparing a reactive solid support surface, which method
comprises the steps of:
[0015] a) providing a solid support having a hydrogel coating with
a plurality of binding elements,
[0016] b) coupling a protein resistant compound to the hydrogel via
a first fraction of the binding elements, and
[0017] c) coupling at least one binding agent to the hydrogel via a
second fraction of the binding elements,
[0018] whereby the protein resistant compound and the at least one
binding agent are co-immobilized to the hydrogel.
[0019] In another aspect, the present invention provides the use of
a reactive solid support surface prepared according to the method
aspect above for analysis of an analyte in a sample.
[0020] In one embodiment of this aspect, the analysis comprises
immunogenicity studies.
[0021] In still another aspect, the present invention provides a
protein resistant solid support surface for coupling of binding
agents, comprising a hydrogel layer with a plurality of binding
elements, wherein a selected fraction of the binding elements are
coupled to a protein resistant compound, and the remaining binding
elements are free for coupling of one or more binding agents to the
surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a diagram showing rat serum anti-IgE responses
versus time after immunization for vaccinated and control animals,
respectively, on a sensor surface with immobilized IgE.
[0023] FIG. 2 is a diagram showing rat serum anti-IgE responses
versus time after immunization for vaccinated and control animals,
respectively, on a sensor surface with immobilized IgE and methoxy
poly(ethylene glycol) amine at a first surface concentration
thereof.
[0024] FIG. 3 is a diagram showing rat serum anti-IgE responses
versus time after immunization for vaccinated and control animals,
respectively, on a sensor surface with immobilized IgE and methoxy
poly(ethylene glycol) amine at a second surface concentration
thereof.
[0025] FIG. 4 is a diagram showing rat serum anti-IgE responses on
sensor surfaces with immobilized IgE and different surface
concentrations of methoxy poly(ethylene glycol) amine.
DEFINITIONS
[0026] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by a
person skilled in the art related to this invention. Also, the
singular forms "a", "an", and "the" are meant to include plural
reference unless it is stated otherwise.
[0027] The term "hydrogel" is used herein in the sense defined by
Merrill, E. W. et al. (1986), Hydrogels in Medicine and Pharmacy,
Vol. III, Ed. Peppas, N. A., Chapter 1, CRC Press (the disclosure
of which is incorporated by reference herein). As stated therein "a
`hydrogel` presents a surface layer of bound molecules which by
reason of their chemical nature hold a large fraction of water, in
which the molecules are predominantly in an amorphous,
water-solvated state."
[0028] "Array" as used herein generally relates to a linear or
two-dimensional array of discrete regions, each having a finite
area, formed on a continuous surface of a solid support and
supporting one or more binding agents.
[0029] "Solid support" as used herein is meant to comprise any
solid (flexible or rigid) substrate onto which it is desired to
apply one or more binding agents, optionally in the form of an
array. The substrate surface supporting the binding agents may be
the surface of a layer of material different from that of the rest
of the substrate.
[0030] "Activation" as used herein means a modification of a
functional group on a molecule to enable coupling to another
molecule.
[0031] "Binding agent" as used herein means a species that exhibits
a (usually specific) binding activity towards a target molecule.
The binding agent may be a member of a specific binding pair,
including, for instance, polypeptides, such as proteins or
fragments thereof; nucleic acids, e.g., oligonucleotides,
polynucleotides, and the like. The binding agent is often a ligand
or a capture agent.
[0032] "Target molecule" as used herein refers to a molecule,
present in a medium, which is the object of attempted capture.
[0033] "Specific binding pair" (abbreviated "sbp") as used herein
describes a pair of molecules (each being a member of a specific
binding pair) that are naturally derived or synthetically produced.
One of the pair of molecules has a structure (such as an area or
cavity) on its surface that specifically binds to (and is therefore
defined as complementary with) a particular structure (such as a
spatial and polar organisation) of the other molecule, so that the
molecules of the pair have the property of binding specifically to
each other. Examples of types of specific binding pairs (without
any limitation thereto) are antigen-antibody, antibody-hapten,
biotin-avidin, ligand-receptor (e.g., hormone receptor,
peptide-receptor, enzyme-receptor), carbohydrate-protein,
carbohydrate-lipid, lectin-carbohydrate, nucleic acid-nucleic acid
(such as oligonucleotide-oligonucleotide).
[0034] "Capture agent" refers to a species that can be immobilized
to a solid support surface and which can bind to another species,
such as a ligand or a second capture agent.
[0035] "Ligand" as used herein means a molecule that has a known or
unknown affinity for a given analyte. The ligand may be a naturally
occurring molecule or one that has been synthesized.
[0036] "Analyte" as used herein is a molecule, e.g., a
macromolecule, such as a polynucleotide or polypeptide, the
presence, amount, identity and/or interaction kinetics of which are
to be determined. The analyte may also be a small molecule. The
analyte is recognized by a particular ligand forming an
analyte/ligand complex.
[0037] "Antibody" refers to an immunoglobulin which may be natural
or partly or wholly synthetically produced and also includes active
fragments, including Fab antigen-binding fragments, univalent
fragments and bivalent fragments. The term also covers any protein
having a binding domain that is homologous to an immunoglobulin
binding domain. Such proteins can be derived from natural sources,
or partly or wholly synthetically produced. Exemplary antibodies
are the immunoglobulin isotypes and the Fab, Fab', F(ab').sub.2,
scFv, Fv, dAb, and Fd fragments.
[0038] "Sensing surface" as used herein is to be interpreted in a
broad sense and means any surface to which a ligand is or can be
immobilized for detecting an analyte/ligand interaction.
[0039] "Surface sensitive detection technique" refers to a
detection technique where a change in a property of a sensing
surface is measured as being indicative of binding interaction at
the sensing surface. Examples of surface sensitive detection
techniques are given in the detailed description of the invention
below.
DETAILED DESCRIPTION OF THE INVENTION
[0040] As mentioned above, the present invention relates to the
preparation of a solid support surface, such as a sensing surface,
which has immobilized on the surface (i) a binding agent(s) and
(ii) a protein resistant compound, which surface permits specific
binding of, e.g., analyte to the immobilized binding agents while
resisting or preventing binding of non-specifically binding species
to the surface.
[0041] According to the invention, this is achieved by providing a
substrate surface having a hydrogel layer attached thereto, and
coupling the protein resistant compound and the one or more binding
agents to the hydrogel, such that the protein resistant compound
and the binding agents are co-immobilized to the hydrogel, i.e.,
that the two molecular species are intermixed laterally on the
hydrogel. While the coupling of protein resistant compound and
binding agent(s) preferably is performed sequentially in the above
order, it may optionally be effected in the opposite order, i.e.,
first coupling binding agent(s) and then protein resistant
compound, or, possibly, also simultaneously.
[0042] The "protein resistant compound" is basically a
biocompatible organic compound which, after coupling to the
hydrogel, lacks (especially polar) functional or charged groups
that may interact with the protein, or groups that may interact via
hydrophobic interaction. It usually incorporates a hydrogen bond
accepting group or groups, and mostly lacks hydrogen bond donors.
The compound is often a hydrophilic polymer, especially a polymer
having hydrogel-like properties. At least when the protein
resistant compound is a polymer, the protein repelling or shielding
effect is believed to primarily be due to entropy effects,
non-specific protein binding giving rise to an unfavourable entropy
increase. The "protein resistant compound" may optionally be a
mixture of different protein resistant compounds.
[0043] For the sake of simplicity, the term "polymer" is used
herein to include oligomers (usually defined as <100 monomer
units, especially <30 monomer units) as well as higher molecular
polymers.
[0044] The most common example of a protein resistant compound is
poly(ethylene glycol) (PEG), which is a linear, flexible,
hydrophilic and water-soluble polyether, which may have a molecular
weight ranging from about 150 to about 10.sup.7, as well as
derivatives and analogues thereof. Usually, the PEG has been
derivatized to contain termini that bind to the hydrogel, such as,
e.g., with an amine or thiol group. PEG analogues include, for
example, PEG-like polymers where the ether linkages are replaced by
amide-bonds making the polymer more stable. Other examples of
protein resistant compound include dendritic polyglycerol (PG)
derivatives (Siegers, C., et al. Chem. Eur. J. 2004, 10,
2831-2838), and the protein resistant compounds described by
Chapman, R. G., et al., J. Am. Chem. Soc. 2000, 122 8303-8304, such
as, e.g., HN(CH.sub.3)CH.sub.2CON(CH.sub.3).sub.2. Numerous other
protein resistant compounds that may be contemplated for use in the
present invention are known to a person skilled in the art.
[0045] Coupling of the protein resistant compound and binding
agents to the hydrogel is effected through binding elements of the
hydrogel and of the protein resistant compound and the binding
agent(s), respectively. The binding elements are preferably
functional groups to permit the formation of covalent bonds. The
functional groups on the hydrogel to which the binding agents are
coupled are preferably, but not necessarily of the same kind as the
functional groups to which the protein resistant compound is
coupled. Exemplary functional groups include hydroxyl, carboxyl,
amino, aldehyde, carbonyl, epoxy, vinyl and thiol.
[0046] In order to increase the coupling of binding agent, the
protein resistant compound may contain an additional functional
group(s), which after coupling of the protein resistant compound to
the hydrogel is accessible for coupling of binding agent. Many
times it may, however, be preferred that the protein resistant
compound does not contain such additional functional groups, so
that the binding agent is only coupled via the functional groups of
the hydrogel.
[0047] Usually, functional groups of the hydrogel are activated by
an activating agent prior to coupling to the protein resistant
compound and the binding agents. Alternatively, functional groups
of the protein resistant compound and/or the binding agents may be
activated. Methods for activating functional groups are readily
apparent to the skilled person and may be selected from a wide
variety of methods. Exemplary activated functional groups include
for example, reactive ester, hydrazide, thiol, maleimide and a
reactive disulphide-containing derivative. Optionally, the
activation/coupling of protein resistant compound and/or binding
agents may be repeated one or more times. Also the actual coupling
steps may optionally be repeated.
[0048] When activating the functional groups on the surface, it is
often preferred not to activate all the functional groups but only
a selected fraction thereof. This is, e.g., the case when the
hydrogel is a carboxymethyl-modified dextran where, as is known per
se in the art, the residual carboxyl groups will exhibit a negative
charge which assists in attracting a positively charged binding
agent (and ultimately analyte to the surface). Depending on the
type of binding agent, a positive charge thereof may be obtained by
selection of the pH.
[0049] In a preferred embodiment, the protein resistant compound is
bound only to a selected fraction of the activated functional
groups, and the binding agent is then bound to remaining activated
groups, optionally after repeated activation of functional groups
on the surface.
[0050] The hydrogel, which may be natural or synthetic, is
preferably a polysaccharide, such as, e.g., dextran, cellulose,
agarose, carrageenan, alginic acid, starch or derivatives thereof
Preferably, the polysaccharide is dextran or cellulose or a
derivative thereof, such as, e.g., carboxymethylated dextran.
[0051] The thickness of the hydrogel layer may vary within wide
limits, but is usually in the range of from about 30 .ANG. to about
10,000 .ANG., especially from about 30 .ANG. to about 1,000
.ANG..
[0052] Coupling to the hydrogel may also be effected via other
binding elements (coupling groups), such as, e.g., members of
specific binding pairs, one member of a specific binding pair being
conjugated to the hydrogel and the other to the protein resistant
compound and to the binding agent, respectively. For example, the
hydrogel may support avidin or streptavidin, and the protein
resistant compound and the binding agent may be biotinylated.
Alternatively, the hydrogel may support an oligonucleotide and a
complementary oligonucleotide may be conjugated to the protein
resistant compound and the binding agent, respectively. Optionally,
one oligonucleotide duplex may be used for the coupling of the
protein resistant compound and a different oligonucleotide duplex
for the binding agent.
[0053] The binding agent is usually a ligand, which is capable of
recognizing a particular analyte in solution. However, the binding
agent may also be a capture agent capable of binding a ligand or,
optionally, a second capture agent, which in turn binds a
ligand.
[0054] Examples of ligands include, without any limitation thereto,
agonists and antagonists for cell membranes, toxins and venoms,
viral epitopes, antigenic determinants, hormones and hormone
receptors, steroids, peptides, enzymes, substrates, cofactors,
drugs, lectins, sugars, oligonucleotides, oligosaccharides,
proteins, glycoproteins, cells, cellular membranes, organelles,
cellular receptors, vitamins, viral epitopes, and immunoglobulins,
e.g., monoclonal and polyclonal antibodies. Among ligands of
particular interest may be mentioned those mediating a biological
function on binding with a particular analyte(s).
[0055] Analytes that may be assayed for include, without any
restriction thereto, agonists and antagonists for cell membrane
receptors, toxins and venoms, viral epitopes, hormones (e.g.,
opiates, steroids, etc), hormone receptors, peptides, enzymes,
enzyme substrates, cofactors, drugs, lectins, sugars,
oligonucleotides, oligosaccharides, proteins, monoclonal and
polyclonal antibodies, and small molecules.
[0056] The solid support is preferably a rigid structure and may
comprise a substrate having a surface layer of a different
material. Exemplary substrate materials are polymers, such as
(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, or
combinations thereof. A preferred substrate material for many
applications is flat glass. The top layer of the solid support may
be of another material than the rest of the solid support. A
suitable surface for many applications is a metal film, e.g., gold,
silver or aluminum, preferably gold.
[0057] With the procedure of the invention, it is possible to
prepare a reactive solid support surface which, despite having a
relatively high level of immobilized protein resistant compound,
still has a high level of an immobilized binding agent, such as a
ligand. In many situations, it is preferred that the amount of
protein resistant compound bound to the surface is not less than
about 2 ng/mm.sup.2. Likewise it is often preferred that the amount
of immobilized binding agent (ligand) is not less than about 9
ng/mm.sup.2.
[0058] While a reactive solid support surface prepared as described
above may be used for any purpose where it is desired to bind one
or more species to the binding agent(s) while substantially
reducing or preventing non-specific binding, the support surface is
preferably used for analytical purposes, e.g., in assays for
qualitative or quantitative analyte determination. Such a surface
is herein sometimes referred to as a "sensing surface".
[0059] When performing an assay for an analyte in a sample, the
binding agent bound to the surface may be an analyte-specific
ligand, the analyte or an analyte analogue, or a capture agent
which in turn binds an analyte-specific ligand. A variety of assay
formats well-known to the skilled person may be used, the three
assay types briefly described below (with respect to mass
sensing-based detection) being only exemplary.
[0060] In an inhibition type assay, the analyte or an analyte
analogue is immobilized on the sensing surface. A high molecular
weight detecting molecule, such as an antibody, is added to the
sample, and the detecting molecules (in excess concentration) bind
to the analyte. When the sample is passed over the sensing surface,
remaining free detecting molecules in the sample will bind to the
surface, and the detected response is inversely related to the
amount of analyte in the sample.
[0061] In a competition type assay, the analyte or an analyte
analogue is conjugated to a high molecular weight carrier forming a
high molecular weight complex which is added to the sample to
compete with the analyte for the binding to analyte-specific ligand
(e.g., an antibody) immobilized on the sensing surface. The
detected response, which will be almost entirely attributable to
the high molecular weight carrier, is inversely related to the
analyte concentration in the sample.
[0062] In a sandwich assay, the response obtained when analyte
binds to analyte-specific ligand immobilized on the sensing surface
is enhanced by passing over the surface a secondary reagent which
binds specifically to bound analyte. The enhancement may be due to
the secondary or "sandwich" reagent being either a larger molecule
or (less commonly) a molecule that binds in a many-to-one ratio.
The detected response is directly related to the analyte
concentration in the sample.
[0063] The reactive solid support surface may also be used for
studying analyte/ligand interactions at the surface for determining
kinetic parameters for the interaction, such as association and
dissociation rate constants and affinity.
[0064] The reactive solid support surface with immobilized protein
resistant compound and binding agents may also be in the form of an
array, where discrete areas or "spots" support different or the
same binding agents. Optionally, the hydrogel coating is also in
array form, i.e., the coating layer consists of separate hydrogel
patches.
[0065] To benefit from the non-specific binding-resisting surface
of the present invention, the sample is usually based on a
"complex" medium containing non-analyte species which may bind
non-specifically to the surface.
[0066] Such a complex medium upon which the sample is based may be
selected from numerous such media containing one or more analytes
of interest. Exemplary complex media include body fluids, such as
cerebrospinal fluid, saliva, breast milk, urine, bile, whole blood,
blood serum or plasma, tears, homogenized biopsies, as well as
other complex media such as cell culture media, cell lysates, crude
plant extracts, extracted or dissolved food stuffs, liquid food
stuffs, such as beverages (milk, fruit juices, beer etc).
[0067] Depending on the particular complex medium to be analyzed,
the tested sample may be the original sample as taken or a dilution
thereof with a suitable diluent. Generally, the complex medium
content of the sample may range from about 1 to about 100% (v/v),
usually from about 10 to about 100% (v/v), especially from about 30
to about 100% (v/v), for example from about 30 to about 50%
(v/v).
[0068] A particular assay for which the reactive solid support
surface prepared according to the invention may be used is for
immunogenicity studies, immunogenicity being the ability of a
substance to induce an immune response, especially in a mammal,
such as a human being. These studies are usually performed on blood
sera to analyze antibodies therein. Such antibodies may, e.g., be
produced in response to certain drugs, such as protein drugs, and
may give rise to undesired side-effects in the patient which reduce
the efficacy of the drug, shorten the time that the drug remains in
the body, and may lead to allergic reactions. The antibodies
elicited against the drug may also cross-react with autologous
antibodies and cause severe problems. The use of an SPR-biosensor
with a sensor surface having an attached carboxymethyl-modified
dextran hydrogel for determination if an immune response against a
therapeutic agent has occurred is, for example, described in US
2003/0040027 A1 (the disclosure of which is incorporated by
reference herein).
[0069] It is often also of interest to analyze antibodies in blood
sera produced in response to vaccination to determine the success
of the vaccination.
[0070] While, as mentioned above, problems with non-specific
binding may be especially severe with mass-sensing sensors,
analyses and assays performed with the reactive solid support
surfaces prepared according to the present invention may be used
with numerous detecting principles including those relying on the
detection of a label, such as a radiolabel, a chromophore, a
fluorophore, etc, as well as label-free techniques. In many cases,
real time detection systems are preferred, especially those based
on chemical sensor or biosensor technology.
[0071] A biosensor is broadly defined as a device that uses a
component for molecular recognition (for example a layer with
immobilized antibodies) in either direct conjunction with a solid
state physicochemical transducer, or with a mobile carrier
bead/particle being in conjunction with the transducer. While such
sensors are typically based on label-free techniques detecting a
change in mass, refractive index or thickness for the immobilized
layer, there are also biosensors relying on some kind of labelling.
Typical sensors for the purposes of the present invention include,
but are not limited to, mass detection methods, such as optical
methods and piezoelectric or acoustic wave methods, including,
e.g., surface acoustic wave (SAW) and quartz crystal microbalance
(QCM) methods. Representative optical detection methods include
those that detect mass surface concentration, such as
reflection-optical methods, including both external and internal
reflection methods, which may be angle, wavelength, polarization,
or phase resolved, for example evanescent wave ellipsometry and
evanescent wave spectroscopy (EWS, or Internal Reflection
Spectroscopy), both of which 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).
[0072] Biosensor systems based on SPR and other detection
techniques are commercially available today. Exemplary such
SPR-biosensors include the above-mentioned Biacore.RTM.
instruments. A detailed discussion of the technical aspects of the
Biacore.sup.0 instruments and the phenomenon of SPR may be found in
U.S. Pat. No. 5,313,264. More detailed information on matrix
coatings for biosensor sensing surfaces is given in, for example,
U.S. Pat. Nos. 5,242,828 and 5,436,161. In addition, a detailed
discussion of the technical aspects of the biosensor chips used in
connection with the Biacore.RTM. instrument may be found in U.S.
Pat. No. 5,492,840. The full disclosures of the above-mentioned
U.S. patents are incorporated by reference herein.
[0073] A sensor chip frequently used in the Biacore.RTM.
instruments has a gold-coated surface with a covalently linked
carboxymethyl-modified dextran polymer hydrogel. The protein
resistant compound, such as poly(ethylene glycol) (PEG) chains with
binding termini, and binding agents, below referred to as
"ligands", may be covalently coupled to such a sensor chip in
several ways.
[0074] In "amine coupling", carboxyl groups of the modified dextran
matrix are activated by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) to give reactive succinimide esters,
which then react spontaneously with amine and other nucleophilic
groups, allowing direct immobilization of ligands containing such
groups. Other groups can be introduced onto the dextran matrix once
it has been activated with EDC/NHS. One example is the introduction
of reactive disulfides that can be used in a thiol-disulfide
exchange reaction to immobilize thiol-containing ligands. Another
example is the introduction of hydrazide groups which can react
with cis-diols obtained by aldehyde-containing molecules.
[0075] "Thiol-coupling" utilizes exchange reactions between thiols
and active disulfide groups. The active disulfide may be introduced
either on the dextran matrix to exchange with a thiol group on the
ligand (ligand thiol approach), or on the ligand molecule to
exchange with a thiol group introduced on the dextran matrix
(surface thiol approach). A common reagent for introducing active
disulfide groups is 2-(2-pyridinylthio)-ethaneamine (PDEA). An
alternative approach is reaction of thiol groups on the ligand with
maleimide reagents introduced on the dextran matrix.
[0076] In "aldehyde coupling", ligands containing aldehyde groups
(either native or introduced by oxidation of cis-diols, e.g., using
sodium metaperiodate) can be immobilized after activating the
surface with hydrazine or carbohydrazide.
[0077] In the following Example, various aspects of the present
invention are disclosed more specifically for purposes of
illustration and not limitation.
EXAMPLE
Instrumentation
[0078] A Biacore.RTM. 3000 (Biacore AB, Uppsala, Sweden) was used.
This instrument, which is based on surface plasmon resonance (SPR)
detection at a gold surface, uses a micro-fluidic system for
passing samples and running buffer through four individually
detected flow cells, designated Fc1 to Fc4, one by one or in
series. As sensor chip was used Series CM5 (Biacore AB, Uppsala,
Sweden) which has a gold-coated (about 50 nm) surface with a
covalently linked hydrogel matrix (about 100 nm) of
carboxymethyl-modified dextran polymer. As running buffer was used
HBS-EP (Biacore AB). Unless indicated otherwise, the flow rate was
5 .mu.l/min. 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.
Rat Sera
[0079] In the experiments below were used sera from rats (Wistar F)
which had and had not, respectively, been vaccinated against
allergy to elicit anti-IgE antibodies (Resistentia Pharmaceuticals
AB, Uppsala, Sweden). The vaccine was a histidine-tagged
recombinant protein, called his-ORO, containing the
receptor-binding domain from rat IgE flanked by the same domain
from opossum IgE (Opossum-Rat-Opossum). The rats were vaccinated
three times with 20 or 100 .mu.g of his-ORO. Control rats were
injected with PBS (phosphate buffered saline) instead of
vaccine.
Optimization of Ligand (IgE) Concentration
[0080] A CM5 sensor chip was activated for 7 minutes with 0.4 M
aqueous 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and
0.1 M aqueous N-hydroxysuccinimide (NHS). Different concentrations
of rat IgE (Zymed Laboratories, Inc., South San Fransisco, Calif.,
U.S.A.) in 10 mM Na-acetate, pH 5.5, were then injected into flow
cells Fc1 to Fc4 of the Biacore.RTM. 3000. The chip surface was
then deactivated for 7 min with 1 M ethanolamine-HCl, pH 8.5. The
activity of the surface was verified by injection of mouse anti-rat
IgE (MARE) monoclonal antibody (Serotec Ltd., Kidlington, U.K.)
(analyte) at 20 .mu.g/ml. Regeneration of the surface was performed
with 10 mM glycine, pH 2.0. The results are shown in Table I below.
TABLE-US-00001 TABLE I Rat IgE conc. Immobilized Analyte response
Flow cell .mu.g/ml (RU) (RU) Fc1 5 8829 2525 Fc2 10 15271 2827 Fc3
20 16504 2680 Fc4 30 16539 2582
[0081] From the table it is seen that a plateau is reached at about
10 .mu.g/ml of rat IgE which would provide robust coupling
conditions, and this concentration was therefore selected for the
experiments below. The level of immobilized IgE to be used was,
however, selected to be about 9000 RU.
Immobilization of PEG and IgE
[0082] Activation of a CM5 sensor chip with EDC/NHS in flow cells
Fc1 to Fc3 was performed for 7 min as described above. 5 mM
methoxy-poly(ethylene glycol) amine (methoxy-PEG-amine;
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--NH.sub.2),
MW 5000 Da (Shearwater Polymers, Inc., Huntsville, Ala., U.S.A.) in
borate buffer (10 mM Na-borate, 1 M NaCl, pH 8.5) were then
injected through flow cells Fc1 and Fc2 for 7 min. To increase the
amount of immobilized PEG in Fc2, the activation was repeated and
10 mM methoxy-PEG-amine were injected for 7 min. The sensor chip
was then left over-night at stand-by flow, after which the surface
was activated again followed by injection of 10 .mu.g/ml of rat IgE
(Zymed Laboratories, Inc.,) in 10 mM Na-acetate, pH 4.5, through
Fc1 to Fc3. The "Aim for ligand level" wizard of the instrument
control software was used to obtain an IgE immobilization level of
about 9000 RU. Deactivation was then performed for 7 min with 1 M
ethanolamine-HCl, pH 8.5.
[0083] The immobilization levels of PEG and IgE obtained in flow
cells Fc1 to Fc3 were as follows:
[0084] Fc1: 1107 RU PEG, 9557 RU IgE
[0085] Fc2: 2301 RU PEG, 8265 RU IgE
[0086] Fc3: 8150 RU IgE.
Analysis of Rat Sera
[0087] Six negative rat sera (serum nos. 4, 11, 28, 38, 49, 58) and
six positive rat sera (serum nos. 20, 25, 39, 45, 51) were taken at
week -1, 5, 7, 9, 11 and 14 after vaccination of the animals (week
-1 being 1 week before vaccination). The sera were diluted 5 times
in HBS-EP (Biacore AB) and analyzed using the CM5 chip immobilized
with PEG and IgE as obtained above. The sera were injected serially
through the flow cells using the "kinject" command, and the
respective responses were determined. 20 .mu.g/ml of MARE (Serotec
Ltd.) was used as control. Regeneration of the surface between each
serum injection was performed with 10 mM glycine, pH 2.25, for 1
minute and 10 mM NaOH for 30 seconds. The "extraclean" command was
used after each regeneration. The results are shown in FIGS. 1 to
3.
[0088] The limit of detection (LOD) was calculated as:
AVERAGE.sub.negative samples+3*SDnegative samples (SD=standard
deviation)
[0089] Samples having responses higher than LOD are therefore
positive with a probability of 99.9%. To conveniently determine
which samples can be detected as positive, the response (in RU) is
divided by LOD. A ratio higher than I thereby indicates a positive
sample.
[0090] The responses for the vaccinated animals were divided by LOD
calculated for all 6 unvaccinated animals at all six times, and the
results are presented in Tables II to IV below. Samples that can be
detected as positive are marked in bold type. (Two samples were
missing, animal 29 week -1 and animal 20 week 9.) TABLE-US-00002
TABLE II 8150 RU IgE RESPONSE/LOD Serum Serum Serum Serum Serum
Serum Week 20 25 29 39 45 51 -1 0.14 0.22 0.18 0.13 0.32 5 0.49
0.66 0.78 0.96 0.58 0.32 7 0.51 0.43 0.81 1.00 1.15 0.34 9 0.64
0.73 0.80 1.20 0.96 11 0.88 0.81 0.79 1.07 1.24 2.32 14 1.02 1.00
0.62 0.76 1.05 2.65
[0091] TABLE-US-00003 TABLE III 1107 RU PEG + 9557 RU IgE
RESPONSE/LOD Serum Serum Serum Serum Serum Serum Week 20 25 29 39
45 51 -1 0.28 0.28 0.47 0.28 0.38 5 0.68 0.47 1.91 2.29 0.61 0.43 7
0.69 0.41 1.95 2.33 1.07 0.66 9 0.94 1.61 1.76 1.63 2.71 11 1.26
1.34 1.46 2.22 1.81 7.02 14 1.37 1.85 1.01 1.54 1.49 8.33
[0092] TABLE-US-00004 TABLE IV 2301 RU PEG + 8265 RU IgE
RESPONSE/LOD Serum Serum Serum Serum Serum Serum Week 20 25 29 39
45 51 -1 0.39 0.31 0.40 0.32 0.29 5 0.62 0.44 2.05 2.13 0.51 0.41 7
0.61 0.38 2.10 2.10 1.02 0.79 9 1.10 1.77 1.52 1.64 3.62 11 1.08
1.60 1.58 1.74 1.94 9.15 14 1.04 2.30 1.06 1.10 1.51 10.83
[0093] As can be seen from FIGS. 1 to 3, 9-14 weeks from
vaccination, sera from vaccinated animals had anti-IgE responses of
249 to 1032 RU on an "IgE surface", and sera from control animals
had 97-385 RU, i.e., a poor separation between vaccinated and
control animals. The corresponding samples on the "2301 RU PEG+8265
RU IgE" surface gave 32-330 RU for vaccinated animals and 8-13 RU
for controls. The high PEG level (2301 RU) is better than the lower
one (1107 RU). As is shown in Tables II to IV, only 10 of 29
potentially positive samples are detected without PEG, whereas 22
of 29 samples are detected as positive with the high PEG level.
Optimization of the Amount of PEG
[0094] The results at week 9 for (vaccinated) serum nos. 25, 29,
39, 45 and 51 from Tables I to IV above are put together in FIG. 4
and in Table V below. In FIG. 4, "X" designates a positive serum
and ".box-solid." designates a negative serum. TABLE-US-00005 TABLE
V Serum 0 RU PEG 1107 RU PEG 2301 RU PEG (vaccinated) response/LOD
response/LOD response/LOD Serum 25 0.64 0.94 1.10 Serum 29 0.73
1.61 1.77 Serum 39 0.80 1.76 1.52 Serum 45 1.20 1.63 1.64 Serum 51
0.96 2.71 3.62
[0095] From Table V and FIG. 4, it is clearly seen that more
samples can be detected as positive, the more PEG there is on the
surface, despite the fact that the response levels for the positive
samples have been considerably reduced.
[0096] 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.
* * * * *