Note: Descriptions are shown in the official language in which they were submitted.
CA 02338269 2001-O1-19
WO 00/05316 1 PCT/DK99/00407
COATING OF SOLID SURFACES WITH ACTIVATED POLYHYDROXYPOLYMERS
FIELD OF THE INVENTION
The present invention relates to a convenient method for coating activated
polyhydroxy-
polymers, e.g. tresyl or maleimido activated dextran, onto solid surfaces.
BACKGROUND OF THE INVENTION
Modification of the physiochemical properties of solid surfaces by
immobilising hydro-
philic chemical entities thereto is known in the art. Conventional methods,
e.g., include
the use of hydrophobic groups or charged groups in order to facilitate the
adsorption of a
hydrophilic entity to a generally hydrophobic solid surface.
The present applicant's earlier international patent application, WO 94/03530,
describes
modification of the hydrophilic properties of solid surfaces by treatment
thereof with an
activated polysaccharide. In that modification process, it is required that
the solid surface
is carrying nucleophilic groups, e.g. amino groups or thiol group, in order to
facilitate im-
mobilisation of, e.g., periodate oxidised dextran or tresyl activated dextran
thereto.
EP 596315 A2 describes a method for coating solid surfaces with dialdehyde
starch
comprising a contacting step and a heating step without an intermediate
rinsing step. It is
stated that the dialdehyde starch is nearly irreversibly attached to some
polymers after
the moderate heating step (50°C to 100°Ci. It is however also
mentioned that the dialde-
hyde starch can be rinsed off quite easily if the heating step is omitted.
WO 91 /05817 and WO 90/06954 describe the immobilisation of polysaccharides to
sur-
faces carrying adsorbed polyamines and WO 91 /09877 describes the
immobilisation of,
e.g., a periodate oxidised cellulose ester to a surface carrying amino groups.
WO 92/07706 describes the immobilisation of conjugates of biopolymers and
polyimines
to a solid surface carrying anionic groups capable of reacting with the amino
groups of
the polyimine.
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WO 92/03732 describes the immobilisation of various water-soluble compounds
onto
solid surfaces, where the water-soluble compounds carry hydrophobic groups so
as to
facilitate the adsorption.
BRIEF DESCRIPTION OF THE INVENTION
It has now surprisingly been found that coating of solid surfaces with
polyhydroxy-
polymers can be accomplished by very simple means, i.e. without a mandatory
prior acti-
vation of the surfaces to be coated and without inclusion of, e.g., aldehyde
groups,
amino group or hydrophobic group in the polyhydroxypolymer. It has in
particular been
found that the coating of microtitre plates (e.g. polystyrene microtitre
plates) with acti-
vated polyhydroxypolymers (e.g. activated polysaccharides such as tresyl
activated dex-
tran (TAD1 or maleimido activated dextran (MAD1) can be accomplished without
the need
for prior coating of the surface with a polyamine or a polyirnine and even
without prior
conjugation of the polyhydroxypolymer with polyimines, polyamines, hydrophobic
li
Bands, and the like.
Thus, the present invention provides a method for coating a solid surface with
a water-
soluble activated polyhydroxypolymer, where the solid surface comprises
substantially no
amino groups, imino groups or thiol groups, the method comprising the step of:
a) contacting a coating solution of the activated polyhydroxypolymer in an
aqueous me
dium having a pH in the range of 1.5-10 and/or having ion strength in the
range of
0.1 to 8 with the solid surface so as to obtain a bonding of the activated
polyhy-
droxypolymer to the solid surface;
b? rinsing the solid surface having the activated polyhydroxypolymer attached
thereto
with a rinse solution; and
c) optionally drying the solid surface having the activated polyhydroxypolymer
attached
thereto.
The present invention also relates to post-treatment of the coated surface so
as to con-
vert the functional groups to other functional groups. Furthermore, the
present invention
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relates to the solid surfaces obtained, their use in immobilisation of
biomolecules, as well
as the thus obtained solid surfaces comprising immobilised biomolecules.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1
Binding of a peptide to polystyrene microtitre wells coated with TAD at
different pH va-
lues. The wells coated with TAD at acidic pH bind more peptide than wells
coated at ba-
sic pH. It is not clear if this effect is due to improved adsorption of the
TAD at low pH or
due to hydrolysis of the tresyl groups at high pH.
Figure 2
Chemical specificity of the TAD coated surface. The amino acids which were
protected
on the a-amino group could bind to the tresyl groups with the side chain only.
The TAD
surface showed pronounced preference for lysine and cysteine, i.e. the
chemical speci-
ficity was restricted to amine and thiol.
Figure 3
A B-cell epitope scan using overlapping peptides performed on a TAD coated
microtitre
plate (A) and a conventional microtitre plate (B, MAXISorp). After
immobilizing the pep-
tides (derived from tumor necrosis factor a (TNFa)) on the two different plate
types,
TNFa antiserum was added. Peptides containing B-cell epitopes were then
expected to be
recognized by the antiserum. It is obvious that more peptides were recognized
on the
TAD coated surface than on the conventional plate.
Figure 4
TAD coated on surfaces other than organic polymers. A glass test tube and a
nickel
spatula were treated with TAD as described in Experimental and tested for
ability to bind
the peptide biotin-MP9. The controls (- TAD coating) were a glass test tube
and a nickel
spatula respectively, treated with the coating solvent only. For comparison an
ODaso/cm2
value was calculated. Obviously, only the TAD coated surfaces bind the
peptide.
Figure 5
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The effect of NaCI when coating MAD on a polystyrene microtitre plate
(MAXISorpl. The
presence of NaCI increased the binding of cysteine by approx. 25%. The
presence of
cysteine binding was detected by adding biotin-NHS. The signal from the lysine
as well
as the signal from the control with buffer only was very low. This documents
that the
binding to the MAD surface is thiol specific.
Figure 6
Coating a polystyrene microtitre plate (MAXISorp) with different
concentrations of MAD.
The optimal coating concentration is between 0.25 and 1 mg/ml. Binding of
cysteine de-
creased with decreasing concentration of MAD. The binding of lysine at all MAD
concen-
tration was very low.
Figure 7
Chemical specificity of the MAD coated surface. The amino acids which were
protected
on the a-amino group could bind to the maleimido groups with the side chain
only. The
MAD surface showed pronounced preference for cysteine, i.e. the chemical
specificity
was restricted to thiol.
Figure 8
Generation of a surface with functional carboxylic acid groups. A TAD surface
prepared
as described in Example 1 was reacted with 6-amino hexanoic acid under various
condi-
tions. The activation with EDC and NHS provided a specific conversion of the
tresyl func-
tional groups to carboxylic acid functional groups.
Figure 9
Generation of a surface with functional thiol acid groups. A TAD surface
prepared as de-
scribed in Example 1 was reacted with 2,2'dithio-bislethylamine/. Subsequent
reaction of
the TAD surface and the thiol-modified TAD surface with biotin-maleimide and
biotin-
MP9 showed that the TAD surface was specific for amines and the thiol-modified
surface
was specific for maleimide without any significant cross-specificity.
DETAILED DESCRIPTION OF THE INVENTION
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As described above, the present invention relates to a method for coating
surfaces with
activated polyhydroxypolymers such as activated polysaccharides.
(The polymer)
5 Polyhydroxypolymers include naturally occurring polyhydroxy compounds such
as poly-
saccharides and synthetic polyhydroxy compounds such as synthetic organic
polymers
e.g. polyvinylalcohol and poly(hydroxymethylmethacrylate). An important common
fea-
ture of such compounds is that they are relatively hydrophilic, which is
reflected in a
good water solubility.
Illustrative examples of naturally occurring polyhydroxy compounds are
polysaccharides,
gum xanthan, etc. Illustrative examples of synthetic organic polymers are
polyvinylalco-
hol, poly(hydroxymethylmethacrylate), poly(hydroxyethylmethacrylate),
poly(hydroxypropylmethacrylate), etc. as well as the corresponding copolymers.
The term "polysaccharide" is intended to be used with its normal meaning, i.e.
"a combi-
nation of nine or more monosaccharides, linked together by glycosidic bonds",
cf.
Hawley's Condensed Chemical Dictionary, 11'" ed., Sax and Lewis, eds., Van
Nostrand
Reinhold Co., New York, 1987. Examples of such polysaccharides are dextran
(e.g. Dex-
tran 40, Dextran 70, Dextran 75), agarose, cellulose and starch.
The present invention is considered especially applicable for polysaccharides
and polyvi-
nylalcohol, in particular polysaccharides such as dextran.
The (weight) average molecular weight of the native polyhydroxypolymer in
question (i.e.
before activation) is typically at least 1,000, such as at least 2,000,
preferably in the
range of 2,500-2,000,000, more preferably in the range of 3,000-1,000,000, in
parti-
cular in the range of 5,000-500,000. It has been shown in the examples that
polyhy-
droxypolymers having an average molecular weight in the range of 10,000-
200,000 are
particularly advantageous.
It is important that the solubility of the polyhydroxypolymer is so that
solvation of the
polymer is preferred over non-specific adsorption to the solid surface. Thus,
In order to
exploit the full scope of the present invention, the polyhydroxypolymer is
preferably wa-
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6
ter soluble to an extent of at least 10 mg/ml, preferably at least 25 mg/ml,
such as at
least 50 mg/ml, in particular at least 100 mg/ml, such as at least 150 mg/ml.
It is known
that dextran, even when activated as described herein, fulfils the
requirements with re-
spect to water solubility.
For some of the most interesting polyhydroxypolymers, the ratio between C
(carbon a-
toms) and OH groups (hydroxy groups) of the unactivated polyhydroxypolymers
(i.e. the
native polyhydroxypolymer before activation) is in the range of 1.3 to 2.5,
such as 1.5-
2.3, preferably 1.6-2.1, in particular 1.85-2.05. Without being bound to any
specific
theory, it is believed that such as a C/OH ratio of the unactivated
polyhydroxypolymer
represents a highly advantageous level of hydrophilicity. Polyvinylalcohol and
polysaccha-
rides are examples of polyhydroxypolymers which fulfil this requirement. It is
believed
that the above-mentioned ratio should be roughly the same for the activated
polyhy-
droxypolymer as the activation ratio should be rather low.
The term "native polyhydroxypolymer" and similar terms are intended to mean
the poly-
hydroxypolymer before chemical modification. Thus, in a native polysaccharide
substan-
tially all monosaccharide units are intact and recognisable.
As mentioned above, the polyhydroxypolymers carry functional groups
(activation
groups), which facilitates the anchoring of secondary molecules (e.g.
peptides, proteins,
antibodies, antigens, nucleic acids, etc. (see below)) to the solid surface. A
wide range of
applicable functional groups are known in the art, e.g. tresyl
(trifluoroethylsulphonyl),
maleimido, cyanogenbromide, tosyl (p-toluenesulfonyll, triflyl
(trifluoromethanesulfonyll,
pentafluorobenzenesulfonyl, and vinyl sulphone groups. Preferred examples of
functional
groups within the present invention are tresyl, maleimido, tosyl, triflyl,
pentafluoroben-
zenesulfonyl, and vinylsulphone groups, among which tresyl, maleimido, and
tosyl groups
are particularly relevant.
It is believed to be advantageous that the functional groups of the activated
polyhydroxy-
polymers according to the invention are attached to the polyhydroxypolymer via
a frac-
tion of the hydroxy groups of the native polyhydroxypolymer. Thus, it is
preferred that
the skeleton of the native polyhydroxypolymer is substantially unaffected by
the activa-
tion.
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Hence, it is also believed that aldehyde functionalities, e.g. arising from
periodate oxida-
tion of polysaccharides, may be disadvantageous as functional groups of the
activated
polyhydroxypolymer as oxidation of a diol to two aldehyde groups markedly
reduces the
hydrophilicity of the polyhydroxypolymer. Thus, preferably, substantially no
aldehyde
groups are included in the polyhydroxypolymer other than any (normally masked)
alde-
hyde functionalities of a native polysaccharide. Functional groups should in
particular not
be aldehyde groups arising from treatment of a polysaccharide with excessive
amounts
(i.e. more than 1 mole per mole hydroxy groups in the polysaccharide) of
periodate.
Furthermore, for the reason mentioned before, it is also preferred that other
functional
groups are not attached to the polyhydroxypolymer via carbon atoms arising
from oxida-
tion of diols to two aldehydes. Thus, it is preferred that a polysaccharide
used within the
scope of the present invention is substantially unmodified before activation
with func-
tional groups.
It should be understood from the above, that the functional groups (activation
groups)
are not polymers in themselves as the method according to the invention is
simpler as
known methods where e.g. poly-L-lysine and other (polylamines/(polylimines are
used as
"activation groups" for immobilising a polysaccharide to a solid surface. It
should in par-
ticular be understood that the functional groups are not polyimines such as
polyethylene
imine or polyamines such as poly-L-lysine. Thus, preferably substantially no
amino (pri-
mary, secondary and tertiary aliphatic and aromatic aminesl, imino, ammonium
(aliphatic
and aromatic ammonium groups such as pyridinium groups), and thiol groups
should be
included in the polyhydroxypolymers when used within the present invention. It
should
also be understood that hydrophobic ligands (e.g. phenyl, naphthyl, pyridyl
and pyridone
groups) which may facilitate the immobilisation of a polysaccharide to some
solid sur-
faces in conventional method are not to be construed as "activation groups"
within of
the present invention, and that substantially no groups of this character
should be in-
cluded in the polyhydroxypolymer.
It is however believed that the functional groups are some-how involved in the
adsorption
of the polyhydroxypolymer to the solid surface. This is i.a. illustrated by
the difference in
the optimal conditions for coating of dextran carrying different functional
groups (tresyl
and maleimidol.
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The polyhydroxypolymers are generally prepared by methods known to the person
skilled
in the art.
Tresyl activated polyhydroxypolymers can be prepared using tresyl chloride as
described
for activation of dextran in Example 1 or as described in Gregorius et al., J.
Immunol.
Meth. 181 (1995165-73.
Maleimido activated polyhydroxypolymers can be prepared using p-
rnaleimidophenyl iso-
cyanate as described for activation of dextran in Example 3. Alternatively,
maleimido
groups could be introduced to a polyhydroxypolymer, such as dextran, by
derivatisation
of a tresyl activated polyhydroxypolymer (such as tresyl activated dextran
(TAD)) with a
diamine compound (generally HEN-C~Hz~-NHZ, where n is 1-20, preferably 1-8),
e.g. 1,3-
diaminopropane, in excess and subsequently react the amino groups introduced
in TAD
with reagents such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-
carboxylate
(SMCC), sulfo-succinimidyl 4-IN-maleimidomethyllcyclohexane-1-carboxylate
(sulfo-
SMCC), succinimidyl 4-/p-maleimidophenyl)butyrate (SMPB), sulfo-succinimidyl 4-
(p-
maleimidophenyllbutyrate (sulfo-SMPB), N-y-maleimidobutyryloxy-succinimide
eaten
(GMBS) or N~y-maleimidobutyryloxy-sulfosuccinimide ester. Although the
different rea-
gents and routes for activation formally results in slightly different
maieimide activated
products with respect to the linkage between the maleimide functionality and
the re-
mainder of the parent hydroxy group on which activation is performed, all and
every are
considered as "maleimide activated polyhydroxypolymers".
Tosyl activated polyhydroxypolymers can be prepared using tosyl chloride as
described
for activation of dextran in Example 2. Triflyl and pentafluorobenzenesulfonyl
activated
polyhydroxypolymers are prepared as the tosyl or tresyl activated analogues,
e.g. by u-
sing the corresponding acid chlorides.
Cyanogenbromide activated polyhydroxypolymer can be prepared by reacting the
polyhy-
droxypolymer with cyanogenbromide using conventional methods. The resulting
func-
tional groups are normally cyanate esters with two hydroxy groups of the
polyhydroxy-
polymer.
The degree of activation can be expressed as the ratio between the free
hydroxy groups
and the activation groups (i.e. functionalised hydroxy groups). It is believed
that a ratio
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between the free hydroxy groups of the polyhydroxypolymer and the activation
groups
should be between 250:1 and 4:1 in order to obtain an advantageous balance
between
the hydrophilicity and the reactivity of the polyhydroxypolymer. Preferably
the ratio is be-
tween 100:1 and 6:1, more preferably between 60:1 and 8:1, in particular
between 40:1
and 10:1.
Especially interesting activated polyhydroxypolymers for use in the method
according to
the invention are tresyl, tosyl and maleimido activated polysaccharides,
especially tresyl
activated dextran (TAD), tosyl activated dextran (TosAD), and maleimido
activated dex-
tran (MAD?.
(The solid surface)
The solid surface to which the polyhydroxypolymer is attached can be selected
from a
wide variety of solid surfaces used in the analytical and diagnostic fields,
however the
solid surfaces are generally characterised in the lack of chemical
functionalities (e.g.
amines, imines and thiols) which are believed to facilitate the coating of
surfaces with
activated polyhydroxypolymers in conventional methods. The most important
types of
solid surfaces are those of organic polymers, glasses, ceramics and metals.
Among the organic polymers, polystyrene, polycarbonate, polypropylene,
polyethylene,
polyethyieneglycol terephthalate, polyvinylacetate, polymethylpentene,
polyvinylpyrrolidi-
none, polyacrylonitrile, polymethylmethacrylate and polyvinylchloride are
illustrative ex-
amples, where polystyrene and polycarbonate are especially interesting
examples.
Among the glasses and ceramics, borosilicate glass (Pyrex glass) and soda-lime
glass are
especially relevant examples, e.g. in the form of specimen tubes, vials, and
slides for mi-
croscopy. The surface of the glass may be treated with acid prior to coating.
Among the metal, nickel, iron, copper gold, silver, aluminium and zinc are the
most reie-
want illustrative examples. Such surfaces are normally cleaned before coating
in order to
remove any metal oxides.
Preferably the solid surface is the surface of a polystyrene body, a
polycarbonate body, a
borosilicate glass body, or a soda-lime glass body.
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The body in itself may have a form or may be designed and shaped for the
particular de-
sired use. E.g. the body may be in the form of a sheet, a film, a bead, a
pellet, a disc, a
plate, a ring, a rod, a net, a filter, a tray, a microtitre plate, a stick, or
a multi-bladed
5 stick. Especially interesting bodies to be coated according to the present
invention are
microtitre plates, e.g. polystyrene microtitre plates, sticks and beads.
It is understood that the surface of the body in question is not already
chemically modi-
fled by coating with a compound before use in the present method. In
particular, the sur-
10 face of the body is not carrying amino, imino or thiol groups. The surface
may however
be irradiated so as to modify the chemical and/or physical properties of the
surface (typi-
tally an oxidative process). It has been shown that irradiation is irrelevant
in one case
(tresyl activated dextran) and slightly advantageous in another case
(maleimido activated
dextran).
Particularly interesting examples of solid surfaces are the surfaces of
polystyrene microti-
tre plates, polystyrene beads, polystyrene sticks, polycarbonate microtitre
plates, glass
beads, and glass plates.
fThe method!
As mentioned above, the method according to the present invention includes a
number of
steps, i.e. the contacting step, the rinse step, and the optional drying step.
These steps
will be described in detail in the following:
a1 contacting step
As the activated polyhydroxypolymers are inherently water soluble, the coating
solution
comprising the activated polyhydroxypolymers is preferably an aqueous
solution. Apart
from the polyhydroxy polymers, such an aqueous solution comprises a pH
adjusting
agent and/or a chaotropic agent, and optional one or more auxiliary
components.
Water is preferred as the solvent i.a. for environmental reasons, economic
reasons and
because many organic polymer materials, such as polystyrene, are damaged by
various
organic solvents such as dimethyl formamide and acetone. Furthermore, organic
solvents
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often interfere with physical, non-covalent adsorption to solid phases. It is
thus preferred
that the solvent comprises less than 5% of organic solvents constituents, more
prefer-
ably no organic solvent constituents are included.
The concentration of activated polyhydroxypolymer in the coating solution is
typically in
the range of 0.001 mg/ml to 5 mg/ml, typically in the range of 0.01 mg/mt to 1
mg/ml,
and preferably in the range of 0.1 mg/ml to 0.5 mg/ml.
With respect to the required amount of activated polyhydroxypolymer per area
unit (area
of the surface to be treated), it is believed that in the range of 0.01-500
ug/cm2 will be
suitable in order to obtain a uniform coating, preferably in the range of 0.06-
200 p.g/cmz,
in particular in the range of 0.1-50 ug/cm2, is used.
As mentioned above, the coating solution which is contacted with the solid
surface has a
pH in the range of 1.5-10 and/or a ion strength in the range of 0.1 to 8.
The desired pH value is obtained by using a pH adjusting agent in the solution
of the ac-
tivated polyhydroxypolymer. pH adjusting agents may be acetic acid, ie.g.
0.596 acetic
acid pH 2.6), a citrate/phosphate buffer (e.g. 0.035 M citrate, 0.075 M
phosphate, pH
5.0), phosphate buffered saline (PBS) (e.g. 0.01 M phosphate, 0.15 M NaCI, pH
7.2) or a
carbonate buffer (e.g. 0.1 M carbonate, pH 9.6). When an acidic pH is
desirable, other
simple organic acids, e.g. formic acid, propionic acid, and butanoic acid, in
concentra-
tions in the range of 0.1-10%, trifluoroacetic acid (0.05-596),
trichloroacetic acid (0.05-
596), HCI (0.01-1 M), HZSOa (0.01-1M) may also be used. For pH adjustment of
the
coating solution acetic acid and HCI are very convenient as "left over" will
be removed
by evaporation be evaporation in the drying process.
Thus, the pH of the coating solution is typically in the range of 1.5-10,
preferably in the
range of 2.0-7.5, more preferably in the range of 2.0-5.5. It has been shown
for a tresyl
activated polyhydroxypolymer that equally advantageous products are obtained
within
the pH range of 2.0-5.5.
In the case of coating a solid surface with tresyl activated dextran, it is
clear from Fig. 1
that low pH is preferred. Coating at e.g. pH 8 might also work if the coating
time is re-
duced in order to minimise hydrolysis of the tresyl groups. However, in a
large scale pro-
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12
duction it is inconvenient to have a very narrow time frame, and as tresyl
activated dex-
tran seems to be very stable at e.g. 0.5% acetic acid in water (approx. pH
2.6) this is a
very suitable solvent composition.
The desired ionic strength is obtained by using a chaotropic agent in the
solution of the
activated polyhydroxypolymer. The chaotropic agent must not contain groups
(e.g. thiols
or amines) that react with the reactive sites of the activated
polyhydroxypolymer or have
any adverse effect on adsorption of the activated polyhydroxypolymer to the
solid phase
(as can e.g. be experienced with some detergents). NaCI, from 0.5 M to 4 M in
water
has been used with satisfactory result. Alternatively, guanidinium chloride,
sodium thio-
sulphate, and sodium thiocyanate may be used. Lower concentration can be used
but
there is a tendency to increased variability of the coating with decreasing
concentration
of the chaotropic agent. Thus, the ionic strength of the solution of the
polyhydroxy-
polymer is typically 0.1-8, preferably 0.5-6, more preferably 0.8-5, in
particular 1.2-4.
It should be understood that a pH adjusting agent may be used in conjunction
with a
chaotropic agent so as to fulfil both the pH and the ionic strength
recommendations.
Auxiliary agents may also be included in the coating solution, however,
preferably no
other constituents are included as it is preferred that all ingredients are
efficiently re-
moved at least in the drying step.
The coating time can be from 1 min to over night, e.g. from 3 hours to over
night, but
the coating time appears to be uncritical. in large scale production over
night coating is
often very convenient.
The coating can be performed at temperature ranging from 4°C to
56°C with equally
good results. However, room temperature is very convenient in order to reduce
tempera-
ture caused edge effects in e.g. microtitre plates which otherwise could lead
to variability
in performance between a well in the centre on the microtitre plate and a well
close to
the edge of the microtitre plate. Furthermore, evaporation during incubation
is less or vir-
tually absent at room temperature compared to elevated temperature.
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Preferred conditions are coating over night at room temperature. These
conditions have
proven advantageous for tresyl activated dextran (TAD) as well as for
maleimido acti-
vated dextran (MAD1.
After the prescribed coating time, the coating solution is removed from the
solid surface
or the solid surface is removed from the coating solution, whatever is most
convenient. .
In the case of a microtitre plate, the coating solution is normally decanted
or pipetted off.
It should be understood that the coating solution is normally not allowed to
completely
evaporate, as the performance of the coating is thereby beyond control in that
a fraction
of the polyhydroxypolymer will be attached to the solid surface by weak
passive adsorp-
tion and may not be efficiently rinsed off in the subsequent step.
b) rinse step
The rinse solution is typically an aqueous solution or simply water. When
acidic condi-
tions are applied in the contacting step, an acidic solution is advantageously
used in the
rinse step in order to avoid that unspecific binding of remaining
polyhydroxypolymer or
other components takes place due to a change in pH. The rinse solution should
contain
no components that could react with the reactive sites of the activated
polyhydroxy
polymer (e.g. amino or thiol groups) or interfere with the coating and should
be easy to
remove in the drying process. Preferably, the rinse solution contains only
little salts or
other non-volatile components.
Acetic acid, e.g. 0.596 in water, is very suitable as the rinsing solution in
situation where
26 acidic conditions have been used in the contacting step as it has a low pH
value (approx.
2.6) and is easily removed by evaporation in the drying process. Water is
suitably used
where the contacting step has been performed under chaotropic conditions.
c) drying conditions
After the rinse step, the surface is dried in order to remove the rinsing
solution and other
volatile components, e.g. acetic acid. Drying is important especially for
storage of the
coated surface and can be performed at a temperature in the range 20°C-
56°C, prefer-
ably in the range of 20°C-45°C, with good results. Drying at
around 37°C ensures that a
relatively fast evaporation of residual rinsing solution takes place and is
often more ad-
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14
vantageous for large scale production of e.g. coated microtitre plates than
drying at
56°C. The drying time can be further reduced under reduced pressure.
After drying, the coated surface can be stored for later use, or may be used
shortly after.
When the coated surface is used immediately after preparation, the drying step
may even
be omitted if the rinse solution is compatible with the solutions used in the
desired appli-
cation.
It should be noted that the coated surfaces prepared according to the present
invention
7 0 have an excellent storage stability expressed as a shelf-life of more than
2-3 years.
Thus, it is preferred and also realistic within the present invention that the
stability of the
coated surface is so that the difference in absorbance for the most absorbing
amino acid
in a test for the amino acid side chain specificity (as described for TAD in
example 6)
when tested on an uncoated solid surface and on a similar solid surface coated
with the
activated polyhydroxypolymer in question has decreased with at the most 2696,
prefer-
ably at the most 1596, more preferably a the most 1096, in particular at the
most 596,
after storage at 37°C for one year. Storage is effected at ambient
conditions with respect
to atmospheric pressure and atmospheric composition. It is believed that
especially pre-
ferred coated surfaces obtained according to the method according to the
invention fulfil
these requirements even when stored at 50°C for one year under the same
conditions.
In a preferred embodiment of the present invention the polyhydroxypolymer is a
polysac-
charide, in particular dextran. Especially important functional groups in
connection with
polysaccharides (e.g. dextran) are tresyl, tosyi and maleimido,
Thus, in one preferred embodiment of the present invention, the method
comprises
a) contacting a solution of a tresyl activated polysaccharide in an aqueous
medium ha-
ving a pH in the range of 1.5-7.5 with a polystyrene surface;
b) rinsing the polystyrene surface with a rinse solution; and
c) drying the polystyrene surface coated with the tresyl activated
polysaccharide.
In another preferred embodiment of the present invention, the method comprises
a) contacting a solution of a maleimido activated polysaccharide in an aqueous
medium
having a ionic strength in the range of 0.5-6 with a polystyrene surface;
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b) rinsing the polystyrene surface with a rinse solution; and
c) drying the polystyrene surface coated with the maleimido activated
polysaccharide.
In still another preferred embodiment of the present invention, the method
comprises
5 a) contacting a solution of a tosyl activated polysaccharide in an aqueous
medium ha
ving a pH in the range of 1.5-7.5 with a polystyrene surface;
b) rinsing the polystyrene surface with a rinse solution; and
c) drying the polystyrene surface coated with the tosyl activated
polysaccharide.
10 For the above-mentioned preferred embodiments, the requirements and
recommendations
mentioned above with respect to the solid surface, the coating conditions and
the poly-
hydroxypolymer also apply.
(Uses)
15 The coated solid surfaces carrying an activated polyhydroxypolymer may be
further func
tionalised before their final use or some or all of the functional groups of
the activated
polyhydroxypolymer may be reacted so as to form other functional groups. In
this way,
functional groups with the ability to facilitate the coating of a solid
surface with a poly-
hydroxypolymer may be chosen in the initial process (step a) to c)) and these
functional
groups may afterwards be converted to other functional groups. The tresyl and
tosyl
groups are good examples of such functional groups for the initial process.
As an example, a tresyl activated polyhydroxypolymer may be reacted with an w-
amino
carboxylic acid (generally HzN-C~Hz~-COOH, where n is 1-20, preferably 1-8) so
as to
form an immobilised carboxylic acid functionalised polyhydroxypolymer. This is
illustrated
in Example 10.
In another variant, a tresyl activated polyhydroxypolymer may be reacted with
an a,w-
diamino-alkane igenerally HzN-C~Hz~-NHx, where n is 1-20, preferably 1-8) so
as to form
an immobilised amino tunctionalised polyhydroxypolymer.
In still another variant, a tresyl activated polyhydroxypolymer may be reacted
with a
cystamine or an analogue igenerally HZN-C~Hz~-S-S-C~Hz~-NHz, where n is 1-10,
prefer-
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16
ably 1-4) and subsequently reduced (e.g. with sodium dithionite) so as to form
an immo-
bilised thiol functionalised polyhydroxypolymer. This is illustrated in
Example 1 1.
Furthermore, the formation of the maleimido functionalised polyhydroxypolymer
via the
tosyl activated polyhydroxypolymer described above may also be accomplished
after
coating of the solid surface by reacting an immobilised amino functional
polyhydroxy-
polymer with a maleimide reagent, e.g. a reagent selected from succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-succinimidyl 4-IN-
maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCCI, succinimidyl 4-(p-
maleimido-
phenyllbutyrate (SMPB), sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate
(sulfo-SMPB),
N-y-maleimidobutyryloxy-succinimide ester (GMBS) and N-y-maleimidobutyryloxy-
sulfo-
succinimide ester.
The above-mentioned variants also apply for other functional groups (e.g.
tosyl, triflyl,
the cyanogenbromide adduct, pentafluorobenzenesulfonyl and vinyl sullen) which
are ca-
gable of reacting with amino groups
Thus, in an interesting embodiment of the present invention the method further
com-
prises the subsequent step (step d)) of converting an amino reactive
functionality of the
solid surface coated with the thus activated polyhydroxypolymer to another
functionality
(e.g. selected from carboxylic acid, amino, thiol, and maleimido) by reacting
the amino
reactive functionality with a reagent which comprises an amino group,
preferably a pri-
mary amino group. The amino reactive functionalities may be functionalities
selected
from tresyl, tosyl, cyanogenbromide, triflyl, pentafluorobenzenesulphonyl and
vinyl sul-
phone. The reagents for such an additional step are exemplified above and the
conditions
(e.g. in an aqueous buffered solution) will be known for the person skilled in
the art.
Especially interesting are the cases within the above-mentioned embodiment
a) where the reagent which comprises an amino group has the general formula
H2N-
C~Hz~-COOH, where n is 1-20, preferably 1-8, whereby an immobilised carboxylic
acid
functionalised polyhydroxypolymer is formed,
b) where the reagent which comprises an amino group has the general formula
HZN-
CnHzn-S-S-C~H2n-NHz, where n is 1-10, preferably 1-4, and where the
intermediate
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17
thus formed is subsequently reduced, whereby an immobilised thiol
functionalised
polyhydroxypolymer is formed, and
c) where the reagent which comprises an amino group has the general formula
HzN-
C~Hz~-NHz, where n is 1-20, preferably 1-8, whereby an immobilised amino
function-
alised polyhydroxypolymer is formed.
As it is believed that the coated solid surfaces are novel in themselves, the
present in-
vention also relates to solid surfaces coated with activated
polyhydroxypolymers. Such
solid surface may advantageously be prepared according to the method of the
present
invention, but alternative methods may also apply.
The solid surfaces prepared according to the invention are particularly useful
for immobi-
lising molecules of various origins. Examples of a particularly interesting
group of mole-
cules is biomolecules such as amino acids, oligo- and polypeptides (a special
example is
PNA), proteins, immunoglobulins, haptens, enzymes, antibodies (monoclonal and
polyclo-
nal), antigenes, polysaccharides, oligo- and polynucleotides (nucleic acids
such as RNA
and DNAI. micro-organisms, procaryotic cells, eucaryotic cells, etc. It has
been experi-
enced that tresyl activated polyhydroxypolymers are especially suitable for
the immobili-
sation of relatively short peptides and nucleic acids such as peptides
consisting of 1-50,
or 1-30, amino acids and nucleic acids consisting of 1-30, ar 1-20,
nucleotides.
Thus, the present invention also provides solid surfaces coated with an
activated polyhy-
droxypolymer as described herein, where one or more biomolecules have been
immobi-
lised to said polyhydroxypolymer via at least a fraction of the activation
groups. The bio-
molecules are typically selected from amino acids, oligo- and polypeptides,
proteins, im-
munoglobulins, haptens, enzymes, antibodies, antigenes. polysaccharides, oligo-
and
polynucleotides, micro-organisms, procaryotic cells, eucaryotic cells. In a
particularly in-
teresting embodiment, the polyhydroxypolymer is a polysaccharide (in
particular dextran)
and the biomolecule is selected from peptides (including PNA) consisting of 1-
30 amino
acids and nucleic acids consisting of 1-20 nucleotides. The tresyl group as
activation
group on the polyhydroxypolymer is especially relevant in these instances.
The conditions for immobilisation of such (bio)molecule are known, see e.g.
Hermanson,
Mallia and Smith, Immobilized Affinity Ligand Techniques, Academic Press,
1992, Immo-
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18
bilized Enzymes and Cells, in Methods in Enzymofogy, Vol 135, Mosbach, Ed.,
Academic
Press, 1987, and US 5,516,673.
Thus, the present invention also relates to the use the solid surfaces
obtained or obtain
s able according to the method of the present invention for immobilisation of
biomolecules.
In another very interesting embodiment of the method according to the present
invention,
the resulting solid surface carry two types of activated polyhydroxypolymer so
as to be
able to immobilise a broader range of biomolecules. Alternatively, the
polyhydroxy-
polymer may carry more than one type of functional groups thereby acting as a
di-acti-
vated polyhydroxypolymer. This can be accomplished either by using two
different acti-
vated polyhydroxypolymers in the contacting step or by only partial conversion
of the
functional groups of the polyhydroxypolymer already coated onto the solid
surface.
The invention is further illustrated by the following examples:
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EXAMPLES
If nothing else is noted chemicals were of analytical grade from Riedel de-
Haen, Seelze,
Germany.
A general method to test the peptide binding capacity
The peptide biotin-MP9 (biotin-FAQKEPAFLKEYHLL) was dissolved (0.01 mg/mll in
0.1 M
carbonate buffer, pH 9.6, and 100 p,l was added to the wells to be tested.
After 60 min
incubation the wells were washed with washing buffer (PBS including 0.5 M NaCI
and
196 Triton x-100 (Sigma)). Residual binding sites were blocked using carbonate
buffer
including 196 bovine serum albumin (BSA, Sigmal, 1596 polyethylene glycol
(PEG) 8000
(Sigma), and 10 mM ethanol amine. After washing, the immobilized peptide was
detec-
ted via the biotin group using a streptavidin-horse radish peroxidase
(streptavidin-HRP,
Amersham) conjugate in diluting buffer (washing buffer including 196 BSA) and
o-phenyl-
enediamine dihydrochloride (OPD, Sigma), 1 mg/ml in substrate buffer (citrate
phosphate
buffer, pH 5.0) as the chromogenic substrate.
Example 1. Synthesis of tresyl activated dextran (TAD)
Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the
dex-
tranl, 4.5 g was dissolved in dry N-methyl-pyrrolidinone (NMP, 225 ml) at 90-
92°C with
magnetic stirring. After cooling to 40°C, 2,2,2-trifluoroethanesulfonyl
chloride (tresyl
chloride), 2764 p,l, was added. After 15 min 2020 pl dry pyridine was added
and the
heating was removed. After 60 min stirring at room temperature (RT) the TAD
was pre-
cipitated in 1200 ml cold ethanol. The precipitate was dissolved in 200 ml
0.596 acetic
acid and dialyzed against 3 times 5 I 0.596 acetic acid in a dialysis membrane
with a
molecular cut off on 12000-14000 Da. After dialysis the TAD was freeze dried.
Example 2. Synthesis of tosyl activated dextran (TosAD)
Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the
dex-
tran), 0.8 g was dissolved in dry N-methyl-pyrrolidinone (NMP, 40 ml) at 90-
92°C with
magnetic stirring. After cooling to 60°C p-toluenesulfonyl (tosyl)
chloride, 2.8 g dissolved
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in dry NMP was added. After 1 min 2 ml dry pyridine was added. After 60 min
the pre-
cipitate is harvested by decanting the supernatant and precipitate is washed
using 10 ml
NMP and subsequently using 10 ml ethanol (99.996). The precipitate is
dissolved in 5 ml
water and precipitated using 30 ml ethanol (99.996). Subsequently the
precipitate was
5 dissolved in 5 ml water and freeze dried. The introduction of tosyl to the
dextran could
be detected by UV at 280 nm.
Example 3. Synthesis of maleimido activated dextran (MAD)
Dextran (Sigma, mW 70000, freeze dried from water to remove water bound to the
dex-
10 trap) 100 mg, was dissolved in dry 1-methyl-2-pyrrolidinone (5 ml) at 90-
92°C with
magnetic stirring. After cooling to room temperature p-maleimidophenyl
isocyanate
(PMPI, Bioaffinity Systems, Roscoe, II, USA), 50 mg dissolved in 1 ml dry
dimethyl sulf-
oxide, was added. After over night incubation the product was precipitated
with 20 ml
ethanol (99.996), dissolved in 5 ml water and freeze dried.
1s
Example 4. The effect of pH on coating TAD onto a solid phase
The effect of pH on the direct coating of activated polyhydroxypolymers, such
as TAD,
was examined by dissolving TAD in different buffers covering the pH range from
2.6 to
9.6. TAD was dissolved in 0.596 acetic acid (pH 2.6), citrate/phosphate buffer
(0.035 M
20 citrate, 0.075 M phosphate, pH 5.0), PBS (0.01 M phosphate, 0.15 M NaCI, pH
7.2) or
carbonate buffer (0.1 M carbonate, pH 9.6) to a final concentration of 0.5 mg
TAD/ml
buffer. The solutions were dispensed into the wells of polystyrene microtitre
plates (Poly-
sorp, Nunc, Denmark), 150 ~I/well, and incubated overnight at room
temperature. The
wells were then washed twice using 0.596 acetic acid and dried overnight at
37°C. The
plates were tested as described in "general method to test the peptide binding
capacity"
and the result is shown in Fig 1. Coating at pH 2.6 and 5.0 resulted surfaces
with the
highest peptide binding capacities. Coating at pH 7.2 reduced the signal from
the peptide
binding by approx. 1596. Coating at pH 9.6 resulted in a signal reduction of
approx.
7096. These results demonstrate that coating TAD directly on solid phases
preferably
should take place under acetic conditions, e.g. in 0.596 acetic acid or
alternatively under
neutral conditions.
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Example 5. Coating TAD on solid phases composed of different materials
TAD, 0.5 mg/ml in 0.5% acetic acid, was added to the surface of different
materials
such as a nickel spatula, a glass test tube, a polycarbonate microtitre plate
and a polysty-
rene microtitre plate and incubated aver night at RT. After washing using
0.596 acetic
acid and drying over night at 37°C the TAD coated materials were tested
as described in
"A general method to test the peptide binding capacity". The peptide binding
test was
performed on TAD coated as well as on not coated samples of each material and
the re-
sults is shown in Fig 4. It is obvious the TAD coating makes the materials
tested in this
experiment capable of binding much more peptide than the uncoated materials.
Example 6. Coating with maleimido activated dextran (MAD) and identification
of the
chemical specificity of the MAD coated surface
Binding of cysteine to a microtitre plate coated with different amounts of
MAD. MAD
was added in serial dilutions, starting at 1 mg/ml, to a microtitre plate
(MAXISorp) in 4 M
NaCI in water and incubated 3 hours at RT. After washing with water the plate
was dried
over night at 37°C. Cysteine and lysine was added, 0.01 mg/ml in PBS,
pH 7.2. After 1
hour incubation the plate was washed and biotin-NHS was added, 0.05 mg/ml in
PBS in-
cluding 0.196 Tween 20, pH 7.2. After 1 hour incubation at RT the wells were
washed
with washing buffer and streptavidin-HRP in diluting buffer (washing buffer
including 196
BSA, pH 7.2), 1:1000, was added. Finally OPD, 1 mgJml in substrate buffer was
added.
The reaction was stopped using 2 N HzSOa the wells were read at 490 nm in an
MRX
ELISA reader (Dynex Technologiesl. Fig 6 shows the result and it is clear that
the binding
of cysteine is dependent on the precoating of the surface with MAD.
Furthermore, this
experiment demonstrates that cysteine (circles) binding takes place via the
thiol group, as
no binding was detected when lysine (triangles) was used. The specificity was
also ex-
amined in a similar experiment using cysteine, lysine, glutamic acid and
glycine (Fig 7).
The amino acids were added to the MAD coated microtitre plate in PBS, 0.01
mg/ml, pH
7.2, and 'incubated 1 hour at RT. The detection of immobilized amino acids
were the pre-
formed using biotin-NHS as described above.
Example 7. The affect of high salt ionic strength when coating MAD on a
microtitre plate
MAD was diluted from a stock solution (20 mglml in water) to 0.5 mg/ml in
solutions of
NaCI ranging from 0 to 4 M NaCI and added to a microtitre plate IMAXISorp).
After 3
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hours incubation the plates were washed with water and dried over night at
37°C. Cys-
teine and lysine, 0.01 mg/ml, (or buffer alone as control) were added
dissolved in PBS
and incubated 1 hour at RT. After washing, immobilization of cysteine or
lysine was de-
tected using biotin-NHS as described in example 6. The results are shown in
Fig 5 and
indicate that increased ionic strength when coating MAD results in increased
binding of a
thiol containing molecule, exemplified in this experiment by cysteine.
Example 8. Testing the amino acid side chain specificity of a TAD coated
surface
An array of amino acids all with a t-butoxycarbonyl (Boc) on the a-amino group
were
added to the wells of a TAD coated polystyrene microtitre plate and incubated
for 1 hour
at RT. After washing the wells with water, the Boc group was stripped off the
immobi-
lized amino acids by treatment with 9596 trifluoroacetic acid (TFA) in water
(black bars in
Fig 2), or treated with water only for control (white bars in Fig 2) 30 min at
RT. The
wells were washed with water and biotin-N-hydroxysuccinimide (biotin-NHS) was
added,
0.05 mg/ml in PBS including 0.196 Tween 20, pH 7.2. After 1 hour incubation at
RT the
wells were washed with washing buffer and streptavidin-HRP in diluting buffer
(washing
buffer including 1 °6 BSA, pH 7.2), 1:1000, was added. Finally OPD, 1
mg/ml in sub-
strate buffer was added. The reaction was stopped using 2 N HzS04 the wells
were read
at 490 nm in an MRX ELISA reader (Dynex Technologiesl. From Fig 2 it is
obvious the
TAD coated surface has a preference for lysine and cysteine side chains, i.e.
primary
amine and thiol. This is in accordance with the theory.
Example 9. Identification of B-cell epitopes using peptides covalently bound
to a TAD
coated microtitre plate
Peptides covering then entire sequence of murine tumor necrosis factor a
(mTNFa) were
synthesised as 15-mer peptides with a 5 amino acid overlap. These peptides
were immo-
bilised (0.05 mg/ml in carbonate buffer, 2 hours at RT) on a TAD coated
microtitre plate
and on a conventional microtitre plate of the high binder type (MAXISorpl.
Subsequently,
after washing and blocking las described in the "A general method to test the
peptide
binding capacity"1, anti-TNFa anti-serum was added. Peptides containing B-cell
epitopes
were then expected to be recognised by the anti-serum. Fig 3 shows the result
of this B-
cell epitope identification assay. More of the covalently immobilised peptides
(A) were
recognised than of the non-covalently immobilised peptides (B). Thus, if the
experiment
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23
had been performed on the conventional plate only and not on the TAD coated
plate, in-
formation about B-cell epitopes in the N-terminal part of the TNFa molecule
would not
have been obtained. The part of the experiment performed on the conventional
plate (B)
did not give any information that could not be obtained from the TAD coated
plate IA).
Example 10. Generation of a surface with functional carboxylic groups using a
TAD
coated surface as platform
To a TAD coated microtitre plate 6-amino hexanoic acid (Sigma), 1 mg/ml in
carbonate
buffer, was added and incubated 2 hours at RT. The plate was then washed and
the
presence of functional carboxylic groups on the surface was tested by
activation of the
carboxylic groups and subsequent binding of a labelled peptide. The carboxylic
groups
were activated by adding fresh solutions of 1-(3-diaminopropyl)-3-
ethylcarbodiimide
(EDC, 0.18 mg/ml in water) and N-hydroxy succinimide (NHS 1.23 mg/ml in
water). As
control, EDC and NHS were either added together, alone or only water was added
and
incubated for 30 min at RT. After washing with water the biotinylated peptide
(biotin-
FAQKEPAFLKEYHLL) was added, 0.01 mg/ml, in PBS including 0.296 Tween 20. The
re-
suit is seen in Fig 8. Clearly, only when both EDC and NHS was used for
activation a
proper binding of the peptide was seen. EDC alone also generates a reactive
ester with a
carboxylic group but it is very unstable in water. NHS alone cannot form a
reactive pro-
duct but if EDC and NHS is present at the same time, the EDC ester reacts
rapidly with
NHS and forms a stable NHS ester.
Example 11. Generation of a surface with functional thiol groups using a TAD
coated sur-
face as platform
To a TAD coated microtitre plate 2,2'-dithio-bis(ethylamine) (cystamine,
Sigma), 1 mg/ml
in carbonate buffer, was added and incubated 2 hours at RT. After washing with
water,
sodium dithionite (Sigma), 2 mg/ml in water, was added and incubated for 3
hours at RT.
The plate was washed with water and the generated thiol groups were detected
by add-
ing biotin-maleimide (Sigma, 0.05 mglml in PBS) which specifically reacts with
thiol
groups, and incubate 1 hour at RT. To test for residual amine binding capacity
(residual
tresyl groups) the peptide biotin-MP9 (biotin- FAC).KEPAFLKEYHLL) was added,
0.01
mg/ml in carbonate buffer, pH 9.6. The immobilized biotin groups (from either
biotin-MP9
or biotin-maleimide) were detected using a streptavidin-horse radish
peroxidase Istrept-
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24
avidin-HRP, Amersham) conjugate in diluting buffer (washing buffer including
196 BSA)
and o-phenylenediamine dihydrochloride (OPD, Sigrnal, 1 mg/ml in substrate
buffer (ci-
trate phosphate buffer, pH 5.0) as the chromogenic substrate. Fig 9 shows how
the TAD
coated surface bound the biotin-maleimide (TAD/biotin-mal) or the peptide
biotin-MP9
(TAD/biotin-MP9) in the two first bars. The third bar is binding of the
maleimide group to
the thiol enriched surface (Thio/biotin-mal) and the last bar is binding of
biotin-MP9
(Thio/biotin-MP9) to the thiol enriched surface. It is obvious the TAD coated
surface by
treatment with cystamine and sodium dithionite lost its ability to bind the
peptide biotin-
MP9 but gained the ability to bind a thiol specific reagent as biotin-
maleimide.
