Note: Descriptions are shown in the official language in which they were submitted.
C~20057~4
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MONOCLONAL ANTIBODIES WHICH RECOGNISE
POLYSULPHATED POLYACCHARIDES
Te~hnical Field
The present invention relates to the preparation of
monoclonal antibodies (MAb) against polysulphated
polysaccharides which contain 2,3-, 2,6- or 4,6- disulphate
ester pyranose ring substitution, to their use in the
detection and/or quantification of polysulphated
polysaccharides which contain 2,3-, 2,6- or 4,6- disulphate
ester pyranose ring substitution in biological samples and
to diagnostic kits for use in the detection and/or
quantification of those polysulphated polysaccharides
comprising the monoclonal antibodies of the invention.
~ckgrolln~ Art
Polysulphated polysaccharides have been shown to
possess a variety of pharmacological activities which in
some instances has led to their development as therapeutic
agents. Heparin, dextran sulphate (DS) and pentosan
polysulphate (PPS) have been widely used as anticoagulants
while an oversulphated chondroitin (GAGPS, Arteparon) has
demonstrated effectiveness in the treatment of
osteoarthritis (Burkhardt and Ghosh, 1987; Rejholec, 1987).
Dextran sulphates have also been used for many years as
antilipemic agents (Ninomiya ~ al, 1988) but more recently
attention has focused on the ability of a low molecular
weight fraction (MW 8000) to block the binding of certain
virions to T lymphocytes (Baba et al, 1988a; Chang et al,
1988; Mitsuya et al, 1988). This latter activity, has also
7581S
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been exhibited by PPS (Baba et al, 1988c) and is currently
being explored clinically as a therapeutic means of
suppressing the effects of HIV-l and HIV-2 viruses which are
responsible for acquired immune deficiency syndrome (AIDS)
in man.
All these polysulphated polysaccharides are strongly
anionic and show charge similarities to heparin and other
naturally occurring sulphated polysaccharides. Their
quantification in biological samples using traditional
colorimetric reagents, such as dimethylmethylene blue
(Farndale et al, 1986), is therefore difficult unless
techniques are employed to remove the interfering endogenous
sulphated polysaccharides which can cause errors in levels
of exogenous polysulphated polysaccharides detected. While
the mono- and disulphated chondroitins have been
successfully resolved and quantified as their
~-unsaturated disaccharides using HPLC (Seldin ç~ al,
1984) this method cannot be applied to the synthetic
heparinoids such as DS or PPS since they are not cleaved by
the chondroitinase ABC necessary to generate the
~-disaccharides for analysis.
In vitro and n v vo experiments conducted in our
laboratories and elsewhere have shown that PPS and DS
possess a range of biological activities which would warrant
their use as antiarthritic (Andrews e~ al, 1983; Golding and
Ghosh, 1983; Lentini et al, 1983; Smith and Ghosh, 1986;
Hutadilok et ~1, 1988; Francis et al, 1989) and antiviral
(Baba et al, 1988b; Chang et al, 1988; Mitsuya et al, 1988)
7581S
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drugs. A convenient, sensitive but specific assay is
therefore required to allow quanification of these molecules
in biological fluids, particularly blood, as this would
facilitate pharmacokinetic and toxicological evaluation of
these drugs in humans, as well as being of use in monitoring
patient response to drug therapy, and patient compliance.
Polysaccharides are weak immunogens and the
elicitation of antibody production by these
non-proteinaceous molecules is considered unusual. However,
there is some evidence that animals develop auto-antibodies
against certain polysaccharides, such as hyaluronic acid
(Underhill, 1982) and monoclonal antibodies against keratan
sulphate fragments derived from proteoglycans have been
available for several years (Caterson et al, 1985a).
Whether these antibodies recognize the keratan sulphate
epitope alone or in combination with the peptide sequence
attached, has not yet been resolved, although the
oversulphated regions close to the core of the proteoglycans
are considered to be important determinants (Caterson et al,
1985b).
Further antibodies have been raised against heparan
sulphate (Japanese Patent Application No. J63052889) and
chondroitin sulphate-D (Japanese Patent Application
No. J63137695-A). These antibodies are clearly dif~erent
from the antibodies of the present invention.
Another observed problem is that endogenous
polysaccharide can cause interference in the conventional
assays for pentosan polysulphate, or dextran sulpate. There
7581S
I A~005194
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are relatively few methods available for the assessment of
polysulphated polysaccharides in biological samples. PPS
has been the most widely studied and bioassays to determine
thrombin inhibitory capacity (Cash et al, 1978), activated
partial thromboplastin time (APTT) or anti-Xa factor
activity (Yin et al, 1973), have been described. A
competitive binding assay (CBA) for the quantification of
PPS employing a radiometric technique was reported by
MacGregor and co-workers (MacGregor et al, 1985), however
this procedure requires the preparation of 125I-labelled
PPS which, because of the short half-life of this isotope,
has reduced shelf-life and thus limitations with respect to
routine application.
Enzyme-linked immunosorbant assays (ELISA) are now
widely used for the assay of a variety of hormones, drugs,
polysaccharides, proteins, and cell derived products, and
this technique offers many advantages over other methods.
Thus, the development of an antibody suitable for use in an
ELISA assay or polysulphated polysaccharides is highly
desirable.
Further, polsulphated polysaccharides can have
anti-serine protease such as anti-elastase activity. It is
recognised that serine proteases such as elastase are
involved in inflammatory conditions such as arthritis,
destruction of connective tissue and emphysema as well as
other inflammatory and degenerative diseases and viral
infections. Thus compounds with anti-serine protease
activity such as antielastase activity are implicated as
7581S
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therapeutic agents for these conditions.
The present inventors have surprisingly found that
there is a good correlation between antielastase activity
and binding to the monoclonal antibody of the invention for
polysulphated polysaccharides.
Thus, the antibodies of the invention can be of use
in the selection of drugs and suggest that monoclonal
antibodies may be of use in selection of drugs where a
particular conformation and charge density, recognised by a
monoclonal is central to the activity of the drug.
Descri ption of the Inventi or
The present invention relates to the preparation, and
characterisation of specific MAbs against polysulphated
polysaccharides. Enzyme-linked immunosorbent inhibition
assays (ELISIA) were set-up using these antibodies to allow
detection and/or quantification of polysulphated
polysaccharides in human serum or plasma and/or other
biological specimens. Using pentosan polysulphate and
dextran sulphate as examples of the methods developed, we
defined the limits for detection of these molecules, and/or
then applied the assay to determine the levels of pentosan
polysulphate in plasma samples of three healthy volunteers,
30 minutes, 1 hour and 2 hours after IV administration.
The method described here offers selectivity,
sensitivity and convenience for the quantification of
polysulphated polysaccharides in biological fluids. The
small study of plasma levels of pentosan polysulphate
achieved after i.v. administration of the drug served to
7581S
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illustrate this point, for the results obtained using our
assay compared favourably with reports by others using
radioactivity-labelled pentosan polysulphate (MacGregor
et al 1984) or a competive binding assay (MacGregor et al
1985). Furthermore, since our MAb only recognised pentosan
polysulphate, its desulpated metabolite xylan, which appears
in the circulation within one to two hours of drug
administration (MacGregor et al 1984), was not falsely
included during the quantification of the drug levels.
The techniques described here thus offer a means of
routinely detecting and/or quantifying polysulphated
polysaccharides which contain 2,3-, 2,6- or 4,6- disulphate
ester pyranose ring substitution in biological samples and
the MAbs when coupled with fluorescent, radioactive
materials or chromogens could be used to visualise or
localise the presence of polysulphated polysaccharides which
contain 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution in histological sections or cell preparations.
According to a first embodiment of this invention
there is provided a monoclonal antibody against a
polysulphated polysaccharide containing 2,3-, 2,6- or 4,6-
disulphate ester pyranose ring substitution.
Typically the monoclonal, antibody recognises
pentosan polysulphate, glycosaminoglycan polysulphates,
dextran sulphate, carrageenan and chondroitin sulphate E,
but does not significantly react with heparin, heparan
sulphate, chondroitin sulphate A, B, C or D or keratan
sulphate.
7581S
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A preferred monoclonal antibody of the invention is
MAb 5-B-10.
According to a second embodiment of this invention
there is provided a monoclonal antibody of the first
embodiment in labelled form.
Typically the label is selected from an enzyme, a
radioisotope a, fluorescent label or biotin.
According to a third embodiment of this invention
there is provided a method for the detection and/or
quantification of a polysulphated polysaccharide in a sample
which method comprises:
providing a solid support coated with a polycationic
coating material and with a polysulphated polysaccharide
containing 2,3-, Z,6 or 4,6- disulphate ester pyranose ring
substitution;
providing a sample;
adding a labelled monoclonal antibody of the second
embodiment, to the sample;
applying the labelled monoclonal antibody containing
sample to the solid support;
removing unbound sample; and
detecting the label.
Typically the solid support is selected from PVC,
polystyrene, Sepharose and agarose.
Preferably the solid support is PVC.
The solid support may be formed as a microtitration
plate, tube or bead.
7581S
CA2005 794
g
Typically the polycationic coating material is
poly-L-lysine.
Typically the polysulphated polysaccharide coat is
selected from pentosan polysulphate, dextran sulphate,
glycosaminoglycan polysulphates, carrageenan and chondroitin
sulphate E.
Preferably the polysulphated polysaccharide is
pentosan polysulphate or dextran sulphate.
Preferably the monoclonal antibody is MAb 5-B-10.
Preferably the sample is a serum or plasma sample.
According to a fourth embodiment of this invention
there is provided a method for the detection and/or
quantification of polysulphated polysaccharides containing
2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution in a sample which methods comprises:
providing a solid support coated with a polycationic
coating material and with a polysulphated polysaccharide
containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution;
providing a sample;
adding a monoclonal antibody of the first embodiment
to the sample;
applying the monoclonal antibody containing sample to
the solid support;
removing unbound sample;
applying a labelled antibody reactive with the
monoclonal antibody to the solid support;
removing unbound labelled antibody; and
7581S
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detecting the label.
Typically, the solid support is selected from PVC,
polystyrene, agarose and sepharose.
Preferably the solid support is PVC.
Preferably the polycationic coating material is
poly -L-lysine.
Typically, the polysulphated polysaccharide coat is
selected from pentosan polysulphate, dextran sulphate,
chondroitin sulphate E, carrageenan and glycosaminoglycan
polysulphates .
Preferably the polysulphated polysaccharide coat is
pentosan polysulphate or dextran sulphate.
Preferably the monoclonal antibody is MAb-5-B-10.
Typically the labelled antibody is an anti-mouse
antibody, preferably an anti-mouse IgM antibody.
Typically the label is selected from an enzyme, a
fluorescent label, a radiolabel or biotin.
Preferably the labelled antibody is alkaline
phosphatase conjugated rabbit anti-mouse IgM.
According to a fifth embodiment of this invention
there is provided a diagnostic kit for use in the detection
and/or quantification of polysulphated polysaccharides
containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution which kit comprises:
a solid support;
a polycationic coating material;
a polysulphated polysacharide coating material
containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
758lS
11 CA2005 794
substitution; and
a labelled monoclonal antibody of the second
embodiment.
Typically the solid support is selected from PVC,
polystyrene, agarose and sepharose.
Preferably, the solid support is PVC.
Typically the polysulphated polysaccharide coating is
selected from pentosan polysulphate, dextran sulphate,
chondroitin sulphate E, carrageenan and glycosaminoglycan
polysulphates.
Preferably the polysulphated polysaccharide coating
is pentosan polysulphate or dextran sulphate.
Preferably the labelled monoclonal antibody is MAb
5-B-10.
Preferably the label is alkaline phosphatase.
According to a si~th embodiment of the invention
there is provided a diagnostic kit for use in the detection
and/or quantification of polysulphated polysaccharides
containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution which kit comprises:
a solid support;
a polycationic coating material;
a polysulphated polysaccharide coating material
containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution;
a monoclonal an~ibody of the first embodiment; and
a labelled antibody which recognises the monoclonal
antibody.
7581S
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Typically the solid support is selected from PVC,
polystyrene, agarose and sepharose.
Preferably the solid support is PVC.
Typically the polysulphated polysaccharide coating is
selected from pentosan polysulphate, dextran sulphate,
glycosaminoglycan polysulphates, carrageenan and chondroitin
sulphate E.
Preferably the polysulphated polysaccharide coating
is pentosan polysulphate or dextran sulphate.
Preferably the monoclonal antibody is MAb 5-B-10.
Typically the labelled antibody is selected from
anti-mouse immunoglobulin antibodies.
Preferably the labelled antibody is an alkaline
phosphatase labelled rabbit anti-mouse IgM antibody.
In the diagnostic kits of the invention it is
preferred that the solid support is coated with
poly-L-lysine.
It is more preferred that the poly-L-lysine coated
support is provided coated with the polysulphated
polysaccharide.
According to a seventh embodiment of this invention,
there is provided a method for determining whether a
polysulphated polysaccharide is a potential antiarthritic,
anti-inflammatory, anti-degenerative, or anti-viral drug
which method comprises:
determining whether the polysulphated polysaccharide
is recognized by a monoclonal antibody of the first
embodiment.
758lS
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Preferably the monoclonal antibody is MAb 5-B-10.
According to an eighth embodiment of this invention
there is provided a method for the visualisation and/or
localisation of polysulphated polysaccharides containing
2,3-, 2,6- or 4,6- disulphate ester pyranose ring
substitution in tissues and/or histological specimens,
comprising administering a labelled antibody of the second
embodiment to the tissue or specimen.
The present invention will now be described by way of
Example only which is not intended to limit the scope of the
invention in any way.
Brief Description of ~h~ Draw~ngs
Figure 1, shows inhibition curves for the
characterization of MAb 5-B-10 using the enzyme-linked
immunosorbent- inhibition assay (ELISIA).
Figure 2, shows sub-typing of the MAb 5-B-10 by the
ELISA technique employing sub-type specific rabbit
anti-mouse immunoglobulins.
Figure 3 shows (a) elution profile of MAb 5-B-10 on
HPHT-column. Fractions were monitored by OD280 (------) and
antibody activity (...... ) using the ELISA technique,
according to the conditions given in Materials and Methods.
The vertical arrow indicates initiation of the linear
gradient. Fractions encompassed by the horizontal bar were
pooled and used for subseguent experiments.
(b) SDS-polyacrylamide gel electrophoresis of ascitic
fluids and purified MAb under reducing condition. Lanes 2
and 7 (diluted ascitic fluids). Lanes 3 and 6 (ammonium
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CA2005794
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sulphate precipitated ascitic fluids). Lanes 4 and 5
(purified MAb from HPHT-column chromatography). Lanes 1 and
8 (low M.W. marker proteins).
Figure 4, shows the saturation curve of MAb 5-B-10 in
human serum obtained using the ELISA technique and wells
coated with 25~g/ml of pentosan polysulphate.
Figure 5, shows the titration curve of various
concentrations of pentosan polysulphate in PBS, pH 7.4 for
coating wells using the ELISA technique with MAb 5-B-10
dilution of 1:400 in human serum.
Figure 6, shows a typical standard inhibition curve
obtained for the quantification of pentosan polysulphate
(M.W. 5000) in human serum using the ELISIA technique, as
described in the Methods section.
Figure 7, shows a typical standard inhibition curve
obtained for the quantification of dextran sulphate (M.W.
5000) in human serum using ELISIA technique, as described in
the Methods section.
Figure 8, shows the kinetics of clearance of pentosan
polysulphate (lmg/kg) in plasma of human volunteers given
the drug intravenously as measured by the ELISIA technique
using MAb 5-B-10.
Best Method of Carryi~g Qut thQ Tnv~ntion
The present invention provides a method for detection
and/or quantification of polysulphated polysaccharides in
human serum and plasma by developing monoclonal antibodies
that recognise the polysulphated polysaccharides and using
the monoclonals in an ELISIA in the following way:
7581S
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(1) normal human sera containing a polysulphated
polysaccharide is incubated with an appropriate dilution of
a first monoclonal antibody;
(2) this reaction mixture is added to the
polysulphated polysaccharide coated plates or other
mmobilized binding source (eg immunobeads, sepharose or
agarose);
(3) enzyme conjugated antibody to first antibody is
added to the plates;
(4) removal of unbound antibodies by washing and
addition of primary substrate;
(5) the secondary substrate is then added and the
plates incubated in the dark to produce a colour reaction;
and
(6) the absorption at a characteristic wavelength
is determined using a microtitration plate reader.
The preferred concentration of human sera or other
biological fluids containing the polysulphated
polysaccharide is 20 - 10,000 ng/ml. It is preferable that
this serum be incubated with an equal volume of its
monoclonal antibody in polypropylene tubes at room
temperature for approximately one hour.
The preferred but not exclusive primary substrate is
NADP in a diethanolamine buffer. The preferred but not
exclusive secondary substrate is alcohol dehydrogenase
diaphorase at 2.1 - 2.4 u/ml; p-iodonitrotetrazolium violet
absolute ethanol all dissolved in phosphate buffer pH 7.2.
7581S
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It is preferable to stop the reaction by the addition
of HCl.
E~ample 1: Materials and Methods
Iscove's modified Dulbecco's medium was obtained from
Gibco Laboratories, Grand Island, N.Y., U.S.A. Freund's
adjuvants were purchased from Difco Laboratories, Detroit,
Michigan, U.S.A. Alkaline phosphatase conjugated rabbit
anti-mouse immunoglobulins were from Dakopatts, Glostrup,
Denmark. Alkaline phosphatase conjugated goat anti-rabbit
IgG, rabbit anti-mouse (subclass specific) immunoglobulin
panel and a high performance hydroxypatite column were
purchased from Bio-Rad Laboratories, Richmond, CA, U.S.A.
Xylan, dextran sulphate (M.W. 5,000), p-nitrophenyl
phosphate, NADP, diaphorase, p-iodonitrotetrazolium violet,
alcohol dehydrogenase, Tween 20 and poly-L-lysine were
obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.
Heparin, heparan sulphate, keratan sulphate, dermatan
sulphate, chondroitin -4- and -6- sulphates were purchased
from Seikagaku Kogyo Co., Ltd., Tokyo, Japan. ~ctivated
polyvinyl chloride immuno-assay plates and a microtitration
plate reader (Twinreader) were obtained from Flow
Laboratories, Zwanenburg, The Netherlands. Pentosan
polysulphate (CartrophenR) was a gift from Arthropharm
Laboratories, Sydney, Australia. ArteparonR was a gift
from Luitpold-Werk, Munich, West Germany.
All other chemicals and reagents were analytical
grade or the highest grade commercially available.
Sodium pentosan polysulphate is the generic chemical
7581S
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name for the polysulphate ester of xylan which is a polymer
prepared semisynthetically from plant raw materials. The
repeating units of xylan polymer are (1--4) linked
~-D-xylopyranoses, with one molecule of the sulphated
esters of a-D-glucopyranosyluronic acid attached to the 2
position of the xylan every nine monomeric sugars. The
degree and positions of substitution of the sulphate esters
and ring conformation of pentosan polysulphate have been
confirmed by 13C-NMR spectroscopy as well as classical
analytical techniques (e.g. sulphur analysis). Like all
polymers, pentosan polysulphate is polydispersed and has a
weight mean molecular weight (Mw) of 5700Da and a number
mean molecular weight (Mn) of 4000 as determined by light
scattering techniques. This would indicate that the major
population consists of 12-16 subunits and therefore n _ 3-4
in the structural formula. The finding of a low Mw/Mn of
approximately 1.5 indicates a level of low polydispersity.
Pentosan polysulphate is a white, odourless, slightly
hygroscopic powder which is readily soluble in water (>20%
at 25). pH(10% w/v H20 - 5.0; n20D (10% w/vH20) ,
1.344; specific rotation [z] D0 ~ _57O (H2O);
sulphated ash , 30%; paper chromatogram (lO~g)Rf . 0.8
(Butanol/H2O; 1:10).
Immunization and Hybr;~oma Development
Female Balb/c mice aged 6-8 weeks were immunized with
polysulphated polysaccharide, e.g. pentosan polysulphate
(lmg/ml in PBS) mixed with an equal volume of complete
Freund's adjuvant. This mixture (50~1) was injected into
7581S
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the footpad of both hind legs. At day 17 post-injection,
cells from the spleen and lymph node of immunized mice were
isolated and were fused with the Balb/c mouse myeloma line
X63-Ag8.653 (a gift from Dr. M. Pollard, Sutton Research
Laboratories, The Royal North Shore Hospital of Sydney)
according to the basic principle described by Kohler and
Milstein (1975) (Galfre and Milsten 1981). Mouse myeloma
line X63-Ag8.653 is described in Kearney et ~1, (1979).
Supernatants from tissue culture media were tested by an
ELISA technique using pentosan polysulphate-coated plates
(see below for details). Hybridomas obtained from positive
wells were cloned and subcloned by the limiting dilution
technique using Balb/c mouse macrophage cells as feeders,
grown in bulk culture. Ascitic fluids were then produced by
injecting the hybridoma cell line intra-peritoneally to
pristane primed Balb/c mice. Monoclonal antibody 5-B-10
specific for the polysulphated polysaccharides, dextran
sulphate and pentosan polysulphate was isolated and
characterised from the ascitic fluid, as described below.
Other monoclonals specific for ArteparonR, heparin and
heparan sulphate were isolated by the same method.
Hybridomas were obtained from the fusion of spleen
cells and myelomas using polyethylene glycol as fusing
agent. Hybridomas from the highest titer of ELISA-positive
wells were expanded and cloned by the limiting dilution
technique. We obtained eight clones which displayed
significant binding to a pentosan polysulphate coated plate
as determined by the ELISA. Clone number 5-B-10 was
7581S
19 CA2~057~4
`
selected from these to produced ascitic fluids in Balb/c
mice. When mice were innoculated with ArteparonR, heparin
or heparan sulphate other clones were obtained which showed
specificity for these sulphated polysaccharides. Here, we
describe as an example the results obtained for 5-B-10. The
diluted ascitic fluid was successfully characterized by
sub-typing of the immunoglobulins by specific antibodies and
the ELISA technique. Hybridoma clone 5-B-10 was found to
produce IgM immunoglobulins and kappa light chains as shown
in Figure 1.
~nzy~e-lin~ mmun~sorbent Assay (~T.TSA) for ~he ~etection
of ~Ah~ ;n Tisslle ~lltl~re Me~;a
An ELISA was used to detect monoclonal antibodies
produced by the hybridomas obtained above. Supernatants
from hybridoma tissue culture plates were added to plates
coated with the respective polysulphated polysaccharide and
incubated at 37C for 90 minutes. The solutions were
removed and the wells then washed 3 times with PBS-Tween
20. Alkaline phosphatase conjugated rabbit anti-mouse
immunoglobulins (1:1000) were then added, the plates were
incubated for 90 mintues, solution aspirated and wells
washed as described above. Alkaline phosphatase substrate
(lmg/ml of p-nitrophenyl phosphate in l.OM diethanolamine
and O.OOlM MgC12, pH 9.8) was added to each well
(100~1/well) and the plates were incubated at 37C
overnight. The release of p-nitrophenyl was monitored by
absorption at 405nm in a microtitration plate reader.
7581S
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.
Antibo~y ~ubclassification
Supernatants from the hybridoma tissue culture media
or diluted ascitic fluids were added to the various
polysulphated polysaccharide coated wells. After incubation
at 37C for 30 minutes, the supernatants were removed and
the wells were washed 3 times with PBS-Tween 20 and 100~1
of rabbit anti-mouse subclass-specific immunoglobulins
(IgGl, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa- and
Lambda-chain) added. After incubation at 37C for 30
minutes, the plates were washed 3 times with PBS-Tween 20.
Alkaline phosphatase conjugated goat anti-rabbit IgG was
added to each well (100~1), the plates were incubated for
30 minutes, and wells washed 3 times with PBS-Tween 20.
Alkaline phosphatase substrate (lmg/ml of p-nitrophenyl
phosphate in 1.0M diethanolamine and 0.001M MgC12) was
then added and the absorption at 405nm which developed at
37C after 60 minutes incubation was determined using a
microtitration plate reader after the reaction was stopped
by the addition of 50~1 of 5N NaOH.
~n7~ e-linked I~llnnsorbant-i~hibition A~say (~T.T~T~) for
Characterization of MonocIo~Al Antibodi~c
Characterization of the monoclonal antibodies (MAbs)
obtained from the hybridomas was performed by using an
ELISIA technique. Various concentrations of inhibitors
(pentosan polysulphate, xylan, dextran sulphate, heparin and
ArteparonR) were incubated with appropriate dilutions of
MAbs at room temperature for 1 hour. The reacton mixtures
were applied to plates in which the polysulphate
7581S
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polysaccharides (e.g. pentosan polysulphate, dextran
sulphate) were bound and the level of MAb which bound to the
wells was determined as described above in the ELISA
method. For this assay, triplicate results were averaged
and the percentage inhibition was calculated. Inhibition
curves were constructed from this data using log/linear
co-ordinates.
Purificat;on of Monoclonal Antibodies ~h~)
Ascitic fluids which were obtained from the Balb/c
mice were diluted, centrifuged (lO,OOOg;10 minutes) and
supernatants applied to a high performance hydroxyapatite
column (HPHT with guard column). Proteins and MAb were
eluted from the column by using a linear gradient of
10-350mM phosphate buffer, pH 6.8 at a flow rate of
lml/minute collecting l.Oml fractions. Fractions were
monitored for absorption at OD280 and tested for antibody
activity by using the ELISA technique as described.
Fractions which contained protein and antibody activity were
pooled and concentrated by ultrafiltration.
Concentrated solutions of the MAbs were run on
SDS-polyacrylamide gel electrophoresis (Phast system;
Pharmacia AB, Uppsala, Sweden) using standard conditions
provided by the manufacturer.
The MAbs were readily purified using the HPHT-column
(Stanker et al, 1985; Juarez-Salinas et ~1 1984) as
demonstrated by the strong binding to pentosan polysulphate
coated-plates (Figure 3A), and the protein distribution on
SDS-PAGE (Figure 3B). The three bands present on SDS-PAGE
7581S
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were identified as the light and heavy chain of IgM of
molecular weight 25,000 and 75,000 daltons, respectively.
The protein band at M.W. 54,000 daltons was identified as
the heavy-chain of endogeneous mouse IgG. This was
confirmed by running this sample on 7.5% SDS-PAGE under
non-reducing condition that showed M.W. about 150,000
daltons (data not shown).
rification of MAh 5-B-10
The elution profile obtained after applying the
ascites fluid to the HPHT-column run as under the condition
described in Materials and Methods section, is shown in
Figures 3A. As is evident, two protein peaks were obtained,
however activity in the ELISA for antibody activity was
confined to the smaller protein peak which eluted in
fractions 47-55. These fractions were pooled, concentrated
and run on SDS-PAGE together with other samples using the
Pharmacia "phast system" containing mercaptoethanol in the
buffer. Figure 3B shows the results obtained after
developing the gel with silver staining. The purified MAb
(Figure 3B tracks 4 & 5) showed 3 bands of molecular weight
25,000, 54,000 and 75,000 dalton.
The MAb clone 5-B-10 was identified as an IgM
immunoglobulin with kappa light chains which would be the
expected response of the animal since the fusion was
achieved just 17 days after the primary immunization by
foot-pad injection of antigen. This particular MAb while
interacting with both pentosan polysulphate and dextran
sulphate did not cross react with heparin, heparin sulphate,
7581S
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ArteparonR, chondroitin sulphates, dermatan sulphate,
keratan sulphate, hyaluronic acid or xylan. However, by
innoculating mice with the appropriate polysulphated
polysaccharide, MAbs specific for heparin, heparin sulphate
and ArteparonR were obtained.
~hAracterization of MAb 5-B-10 by ~T.TST~ Techni~le
Figure 2 shows the inhibition curves obtained using
MAb 5-B-10 in the ELISIA for the polysaccharides pentosan
polysulphate, dextran sulphate, xylan, sodium heparin and
ArteparonR. No interaction was found for dermatan
sulphate, chondroitin -4- or -6- sulphate, keratin sulphate
or hyaluronic acid (data not shown). It is clear that using
MAb 5-B-10, only pentosan polysulphate and dextran sulphate
were inhibitory to the binding of the antibody to pentosan
polysulphate coated wells. The slope of the inhibition
curve and IC50 (concentration at 50% inhibition) between
pentosan polysulphate and dextran sulphate were not
significantly different. Other clones isolated from the
fused hybridoma cells showed selectivity for heparin,
heparan sulphate and ArteparonR, using the same techniques
(data now shown).
Coating Microtitration Plates with Polys~llphAted
Polysaccharides
Activated polyvinyl chloride immuno-assay plates were
pre-coated with poly-L-lysine by the addition of a solution
of 100~1/well of 50~g/ml solution. After incubation for
1 hour at 37C, the solution was flicked out of the wells
and the plates air-dried. One hundred microlitres of a
7581S
24 CA2005794
solution (50~g/ml) in PBS, pH 7.4 of the polysulphated
polysaccharide pentosan polysulphate or dextran sulphate was
pipetted into the poly-L-lysine coated wells. The plates
were then incubated at 37C for 1 hour, the wells were
washed three times with PBS-Tween 20 (0.05% v/v) and 1%
BSA-PBS (100~1/well) was added. A further incubation at
37C for 1 hour followed by 5X wash with PBS-Tween 20 and
air-drying afforded the required polysulphated
polysaccharide coated plates. The coated plates were
wrapped in polythene film and stored at 4C until needed.
~a~ple 2: Amplif;~ ~T.TSTA for ~Antitati o~ of
Polysulph~ted Polysacchari~ in Norma~ man Serum
Pooled normal human sera which containing pentosan
polysulphate (20-lO,OOOng/ml) was incubated with an
appropriate dilution of MAb 5-B-10 (equal volume) in
polypropylene tubes at room temperature for 1 hour. The
reaction mixture was added to pentosan polysulphate coated
plates and processed as described above in the ELISIA
method. After adding alkaline phosphatase conjugated rabbit
anti-mouse immunoglobulins and removing unbound antibodies
by washing, 40~1 of primary substrate (0.2mM NADP in 50mM
diethanolamine buffer pH 9.5) was added to the wells and the
plates were incubated in the dark for 20 minutes. To the
wells was then added 110~1 of secondary substrate (alcohol
dehydrogenase at 0.4 mg/ml; diaphorase at 2.1-2.4U/ml;
p-iodonitrotetrazolium violet at 0.278mg/ml; absolute
ethanol at 4% all dissolved in 25mM phosphate buffer pH 7.2)
and the plates were incubated in the dark at room
7581S
CA2005794
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temperature for 5-10 minutes. The reaction was stopped by
the addition of 25~1 of 0.4M HCl. The absorbance at 492nm
was determined using a microtitration plate reader.
The saturation curve obtained using the various
dilutions of MAb 5-B-10 (or other MAbs) made up in normal
human serum which were incubated in wells of the pentosan
polysulphate coated plates is shown in Figure 4. From this
curve the dilution at 1:400 was selected to be the most
appropriate to be used in the assay. Figure 5 shows the
saturation curve obtained for various concentrations of
pentosan polysulphate used for coating wells at 37C for 1
hour. The concentration of 25~g/ml of pentosan
polysulphate in PBS at pH 7.4 was selected to be the most
appropriate and was used thereafter in the standard assay.
Using the above conditions the quantification of
pentosan polysulphate and dextran sulphate in human serum
was assessed and a typical result is shown in Figure 6 and
7, respectively. The levels of pentosan polysulphate
detected in human volunteers administered l.Omg/kg as a
function of time is shown in Figure 8. The result shows
that this assay can be used to quantify amounts of
polysulphated polysaccharides within the range of
50-5000ng/ml of serum or plasma. The intra- and inter-assay
coefficients of variation were 4.17 + 2.80 and 16.73 +
13.84%, respectively. The percentage recovery of
polysulphated polysaccharides by this method was determined
by adding known amounts of pentosan polysulphate to pool
normal sera and assaying levels by using the standard
7581S
C A200 5 7 94
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procedure. It was found that the precentage of recovery
using our method was 92.65 + 29.85%.
Determi~Ation of Polysulphate Polysacchari~ in ~llm~n
Plasma
Three healthy volunteers were fully informed of the
procedures and aims of the study. Each subject was injected
intravenously with pentosan polysulphate at a dose of lmg/kg
body weight. Blood samples were taken by clean venepuncture
from the anti-cubital veins immediately prior to drug
administration and at 30, 60 and 120 minute intervals.
Blood was collected in tubes containing oxalate and the
plasma was separated by centrifugation and used directly for
the determination of pentosan polysulphate levels. Plasma
taken from each subject prior to drug administration was
used to prepare the standard curve.
Method for Determination of Anti-Elastase Activity
of ~ulphated Polysacchari~
Inhibition of human granulocyte elastase (HGE) by
sulphated polysaccharide was measured using the following
20 assay procedure.
Assay Buffer 50mM sodium phosphate pH 7.4
0.1% (w.v.) BSA (bovine serum albumin)
0.02% (v/v) Triton X100.
Substrate: 0.2mM Succinyl-alanine-alanine-valyl-
nitroanaline (SAAVNA) added as 100~1 of
0.5mM SAAVNA in 12.5% DMSO/87.5% assay
buffer. This gave a final assay
concentration of 5% DMSO
(dimethylsulfoxide).
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-
Inhibitor: Diluted to less than lO~g/ml with assay
buffer. Then assayed as serial dilutions
of this concentration. Added as 100~1 in
assay buffer.
Enzyme: 0.2~g HGE added in 50~1 assay buffer.
100~1 inhibitor and 50~1 enzyme were added to
microtiter plate wells. The plate was incubated at 37C for
5 minutes prior to initiation of the reaction by addition of
prewarmed substrate solution. Absorbances at 405nm were
read at regular intervals and the change in absorbance per
hour calculated. The inhibitor concentration giving 50%
inhibition of enzyme activity compared to an uninhibited
control was calculated (IC50~.
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`_
Correlation of Binding to MAb 5-B-10 and Anti-elastase (HGE)
Activity of Sulphated Polysaccharides
Compound Anti-elastase Binding
Activity (IC50) to MAb 5-B-10
(~g/ml)
Chondroitin-6-sulphate > 10.00
Chondroitin-4-sulphate > 10.00
10 Dermatan sulphate > 10.00
Chondroitin-4,6-disulphate1.87 ++++
Chondroitin-2',6-disulphate > 10.00
Chondroitin polysulphate0.77 ++++
(Arteparon )
Pentosan polysulphate0.88 ++++
Keratan sulphate > 10.00
Dextran sulphate 1.34 ++++
(M.W. 8,000)
Tn~ trial Application~
The monoclonal antibodies of this invention can be used
in the detection and/or quantification of polysulphated
polysaccharides in biological samples, in the methods of the
invention and using the diagnostic kits of the invention.
In labelled form, the antibodies of the invention may
be used to visualise or localise polysulphated
polysaccharides in histological preparations at cell
preparations.
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`_
The antibodies are also of use in the selection o~
potential drugs with anti-arthritc, anti-inflammatory, anti-
degenerative, anti-coagulant and/or anti-viral activities.
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