WO1990006954A1

WO1990006954A1 - Monoclonal antibodies which recognise polysulphated polysaccharides

Info

Publication number: WO1990006954A1
Application number: PCT/AU1989/000547
Authority: WO
Inventors: Peter Ghosh; Prachya Kongtawelert
Original assignee: The University Of Sydney
Priority date: 1988-12-19
Filing date: 1989-12-19
Publication date: 1990-06-28
Also published as: CA2005794A1

Abstract

The present invention relates to monoclonal antibodies which recognise polysulphated polysaccharides which contain 2,3-; 2,6-; or 4,6-disulphate ester pyranose ring substitution. These antibodies are used in assays for the detection and/or quantification of polysulphated polysaccharides which contain 2,3-; 2,6-; or 4,6-disulphate ester pyranose ring substitution in biological fluids. The invention also relates to diagnostic kits for use in the assays.

Description

MONOCLONAL ANTIBODIES WHICH RECOGNISE

POLYSULPHATED POLYSACCHARIDES

Technical 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.

Background 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 fit .al, 1988) but more recently attention has focused on the ability of a low molecular weight fraction (M 8000) to block the binding of certain virions to T lymphocytes (Baba fit al 1988a; Chang fit S , 1988; Mitsuya fit .il, 1988). This latter activity, has also been exhibited by PPS (Baba fit ϋλ, 1988c) and is currently being explored clinically as a therapeutic means of suppressing the effects of HIV-1 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

SUBSTITUTE SHEET colorimetric reagents, such as dimethylmethylene blue (Farndale fit 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 Δ-unsaturated disaccharides using HPLC (Seldin fit 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 Δ-disaccharides for analysis.

In vitro and in vivo 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 ≤t al/ 1983; Golding and Ghosh, 1983; Lentini fit al, 1983; Smith and Ghosh, 1986; Hutadilok fit al, 1988; Francis fit al, 1989) and antiviral (Baba fit al/ 1988b; Chang fit al, 1988; Mitsuya fit al, 1988) 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 fit 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 gt 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 different 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 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 gt al, 1978), activated partial thromboplastin time (APTT) or anti-Xa factor activity (Yin fit 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 fit 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 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.

Description of the Invention 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 illustrate this point, for the results obtained using our assay compared favourably with reports by others using radioactivity-labelled pentosan polysulphate (MacGregor fit al 1984) or a competive binding assay (MacGregor _fit 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 fit 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.

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-, 2,6 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.

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 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 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 sixth 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 antibody of the first embodiment; and a labelled antibody which recognises the monoclonal antibody.

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. 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 the Drawings 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 subsequent 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 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 25μg/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 Carrying Out the Invention 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:

(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. If 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. It is preferable to stop the reaction by the addition of HC1. Example 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. Activated polyvinyl chloride immuno-assay plates and a microtitration plate reader (Twinreader) were obtained from Flow Laboratories, Zwanenburg, The Netherlands. Pentosan polysulphate (Cartrophen ) was a gift from Arthropharm

Laboratories, Sydney, Australia. Arteparon 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 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 β-D-xylopyranoses, with one molecule of the sulphated esters of α-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 H,0 - 5.0; n20°_n (10% w/vH-0) = 1.344; specific rotation [z] _D 20 = -57° (H-O); sulphated ash = 30%; paper chromatogram (10μg)R_f - 0.8

(Butanol/H₂0; 1:10). Immunization and Hvbridoma 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 (50μl) was injected into 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 fit al, (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 hybridorna 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 Arteparon , 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 selected from these to produced ascitic fluids in Balb/c mice. When mice were innoculated with Arteparon , 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.

Enzvme-linked Immunosorbent Assay (ELISA) for the Detection of MAbs in Tissue Culture Media

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 37°C 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 1.0M diethanolamine and 0.001M MgCl₂, pH 9.8) was added to each well

(lOOμl/well) and the plates were incubated at 37°C overnight. The release of p-nitrophenyl was monitored by absorption at 405nm in a microtitration plate reader. Antibody Subclassification Supernatants from the hybridoma tissue culture media or diluted ascitic fluids were added to the various polysulphated polysaccharide coated wells. After incubation at 37°^ for 30 minutes, the supernatants were removed and the wells were washed 3 times with PBS-Tween 20 and lOOμl of rabbit anti-mouse subclass-specific immunoglobulins (IgG₁, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa- and Lambda-chain) added. After incubation at 37°C 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 (lOOμl), 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 MgCl_^) was then added and the absorption at 405nm which developed at 37°C after 60 minutes incubation was determined using a microtitration plate reader after the reaction was stopped by the addition of 50μl of 5N NaOH.

Enzyme-linked Immunosorbant-inhibition Assay (ELISIA) for Characterization of Monoclonal Antibodies

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 Arteparon ) were incubated with appropriate dilutions of MAbs at room temperature for 1 hour. The reacton mixtures were applied to plates in which the polysulphate 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. Purification of Monoclonal Antibodies (MAbs)

Ascitic fluids which were obtained from the Balb/c mice were diluted, centrifuged (10,000g;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 1.0ml 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 fit al/ 1985; Juarez-Salinas fit al 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 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 πon-reducing condition that showed M.W. about 150,000 daltons (data not shown) . Purification of MAb 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, Arteparon , 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 Arteparon were obtained. Characterization of MAb 5-B-10 bv ELISIA Technique

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 Arteparon . 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 Arteparon , using the same techniques (data now shown) .

Coating Microtitration Plates with Polysulphated Polysaccharides

Activated polyvinyl chloride immuno-assay plates were pre-coated with poly-L-lysine by the addition of a solution of lOOμl/well of 50μg/ml solution. After incubation for 1 hour at 37°C, the solution was flicked out of the wells and the plates air-dried. One hundred microlitres of a solution (50μg/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 37°C for 1 hour, the wells were washed three times with PBS-Tween 20 (0.05% v/v) and 1% BSA-PBS (lOOμl/well) was added. A further incubation at 37°C 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 4°C until needed. Example 2: Amplified ELISIA for Ouantitation of Polysulphated Polysaccharides in Normal Human Serum

Pooled normal human sera which containing pentosan polysulphate (20-10,000ng/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, 40μl 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 HOμl 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 temperature for 5-10 minutes. The reaction was stopped by the addition of 25μl of 0.4M HC1. 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 37°C for 1 hour. The concentration of 25μg/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 procedure. It was found that the precentage of recovery using our method was 92.65 + 29.85%.

Determination of Polysulphate Polysaccharides in Human 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 Sulphatefl PQlysacgharides

Inhibition of human granulocyte elastase (HGE) by sulphated polysaccharide was measured using the following 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 lOOμl of 0.5mM SAAVNA in 12.5% DMSO/87.5% assay buffer. This gave a final assay concentration of 5% DMSO (dimethylsulfoxide) . Inhibitor: Diluted to less than lOμg/ml with assay buffer. Then assayed as serial dilutions of this concentration. Added as lOOμl in assay buffer. Enzyme: 0.2μg HGE added in 50μl assay buffer. lOOμl inhibitor and 50μl enzyme were added to microtiter plate wells. The plate was incubated at 37°C 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 (IC_5Q).

Correlation of Binding to MAb 5-B-10 and Anti-elastase (HGE)

Activity of Sulphated Polysaccharides

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.

The antibodies are also of use in the selection of potential drugs with anti-arthritc, anti-inflammatory, anti- degenerative, anti-coagulant and/or anti-viral activities.

REFERENCES

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Baba, M. , Nakajima, M. , Schols, D., Pauwels, R., Balzarini, J. and de Clercq, E. Pentosan polysulfate, a sulfated aligosaccharide, is a potent and selective anti-HIV agent in vitro. Antiviral. Res. 9: 335-343, 1988b. Baba, M. Pauwels, R., Balzamini, J. , Arnaut, J. , Desmyter, J. and de Clercq, E. Mechamism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro. Proc. Nalt. Acad. Sci. USA, 85: 6132-6136, 1988a. Baba, M. Snoeck, R, Pauwels, R. and de Clercq, E. Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus. Antimicrob. Agents Chemother. 32: 1742-1745, 1988c.

Burkhardt, D. and Ghosh, P. Laboratory evaluation of antiarthritic drugs as potential chondroprotective agents. Semin. Arthritis Rheum. 12 (Suppl. 1), 3-34, 1987. Cash, H.D., Gader, A.M.A., Mulder, J.L. and Cort, J.H. Structure activity relations in the fibrinolytic response to vasopressin in man. Clin. Sci. Molec. Med. 5_£, 403-409, 1978. Caterson, B., Baker, J.R., Christner, J.E., Lee, Y. , Lentz, M. Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycans. J. Biol. Chem., Vol. 260(19): 11348-11356, 1985b. Caterson, B., Christner, J.E., Baker, R. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. J. Biol. Chem. 358: 8848-8854, 1985a. Caterson, B., Christner, J.E., Baker, J.R. and Couchman,

J.R. Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. Fed. Proc. 14., 386-393, 1985. Chang, R.S., Tabba, H.D., He, Y.S. and Smith, K.M. Dextran sulfate as an inhibitor against the human immunodeficiency virus. Proc. Soc. Exp. Biol. Med.

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22, 173-184, 1983. Hutadilok, N., Smith, M.M. , Cullis-Hill, D., Brooks, P.M. and Ghosh, P. Pentosan polysulphate stimulates hyaluronate and DNA synthesis in synovial fibroblasts and partially reduces the suppressive effect of hydrocortisone on fibroblast metabolism. Curr. Ther.

Res. 4_4_, 1-16, 1988. Juarez-Salinas, H., Engelhorn, C, Bigbee, W.L., Lowry, M.A. and Stanker, L.H. Ultrapurification of monoclonal antibodies by high-performance hydroxyapatite (HPHT) chromatography. BioTechnique, May-June, 164-169, 1984. Kearney J.F., Radbruch A, Liesegang B and Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunology 123 1548-1551, 1979.

Kohler, G., and Milstein, C. Continous cultures of fused cells secreting antibody of predefined specificity. Nature, 256. 495-497, 1975. Kongtawelert, P., Francis, D.J., Brooks, P.M. and Ghosh, P. The application of enzyme-linked immunosorbent. Inhibition assay to quantitate the release of KS-peptides into fluids of rat subcutaneous air-pouch model and the effects of chondroprotective drugs on this processe. Rheum Int JL 77-83 1989. Lentini, A., Ternai, B. and Ghosh, P. Synthetic inhibitors of human leukocyte elastase. Part 1: Sulphated polysaccharides. Biochem. Inter. 10: 221-232, 1985. MacGregor I.V., Dawes J. , Paton L., Pepper D.S., Prowse C.V. and Smith M. Metabolism of sodium pentosan polysulphate in man-catabolism of iodinated derivatives. Throm Haemostas 5_L, 321-325, 1984. MacGregor, I.v., Dawes J., Paton L, Pepper D.S., Prowse C.V. and Stocks J, . Metabolism of sodium pentosan polysulphate in man measured by a new competitive binding assay for sulphated polysaccharides - comparison with effects upon anticoagulant activity, lipolysis and platelet - granule proteins. Thromb. Haemob. 5_3_, 411-414, 1985. MacGregor, I.V., Dawes, J., Paton, L., Pepper, D.S., Prowse, C.V. and Smith, M. Metabolism of sodium pentosan polysulphate in man - catabolism of the iodinated derivatives. Thromb. Haemostas. 5J, 321-325, 1984. Mitsuya, H., Lonney, D.J. Kuno, S., Ueno, R., Woong-Staal, F. and Broder, S. Dextran sulfate suppression of viruses in the HIV family-inhibition of virion binding to CD4+ cells. Science 240: 646-649, 1988. Ninomiya, K., Saito, F., Mukaida, H., Maruhama, Y. Effect of oral administration of dextran sulphate on endogenous plasma lipoprotein lipase and hepatic-lipase activitiesin healthy men. J. Clin. Biochem. Nutr. 5(1): 59-66,1988.

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Claims

1. A monoclonal antibody against a polysulphated polysaccharide containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring substitution.

2. A monoclonal antibody according to Claim 1 which 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.

3. A monoclonal antibody according to Claim 1 or Claim 2 wherein the antibody is an IgM antibody.

4. A monoclonal antibody according to Claim 3 wherein the antibody has kappa light chains.

5. The monoclonal antibody MAb 5-B-10 as hereinbefore defined.

6. A monoclonal antibody according to any one of Claims 1 to 5 in labelled form.

7. A monoclonal antibody according to Claim 6 wherein the label is selected from an enzyme, a radioisotope, a fluorescent label and biotin.

8. A method for the detection and/or quantification of a polysulphated polysaccharide containing 2,3- 2,6- or 4,6- disulphate ester pyranose ring substitution in a sample which method comprises: providing a solid support coated with a polycatonic 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 labelled monoclonal antibody according to claim 6 or 7 to the sample; applying the labelled monoclonal antibody containing sample to the solid support; removing unbound sample; and detecting the label.

9. The method according to Claim 8 wherein the solid support is selected from PVC, polystyrene, agarose and sepharose.

10. The method according to Claim 9 wherein the solid support is PVC.

11. The method according to any one of Claims 8 to

10 wherein the solid support is formed as a microtitre plate, a tube or a bead.

12. The method according to any one of Claims 8 to 11 wherein the polycationic material is poly-L-lysine.

13. The method according to any one of Claims 8 to

11 wherein the polysulphated polysaccharide coating material is selected from pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphates, carrageenan and chondroitin sulphate E.

14. The method according to Claim 13 wherein the polysulphated polysaccharide is pentosan polysulphate or dextran sulphate.

15. The method according to any one of Claims 8 to 14 wherein the monoclonal antibody is MAb-5-B-10.

16. The method according to any one of Claims 8 to 15 wherein the sample is a serum or plasma sample.

17. A method for the detection and/or quantification of a polysulphated polysaccharide containing 2,3-, 2,6- or 4,6- disulphate ester pyranose ring substitution in a sample, which method comprises: providing a solid support coated with a polycatonic coating material and with a polysulphated polysaccharide containing 2,3-, 2,6- or 4,6- disulphate pyranose ring substitution; providing a sample; adding a monoclonal antibody according to any one of claims 1 to 5 to the sample; applying the monoclonal antibody containing sample to the solid support, removing the unbound sample; applying a labelled antibody reactive with the monoclonal antibody to the solid support; removing unbound labelled antibody and detecting the label.

18. The method according to Claim 17 wherein the solid support is selected from PVC, polystyrene, agarose and sepharose.

19. The method according to Claim 18 wherein the solid support is PVC.

20. The method according to any one of Claims 17 to

19 wherein the polycationic coating material is poly-L-lysine.

21. The method according to any one of Claims 17 to

20 wherein the polysulphated polysaccharide is pentosan polysulphate, dextran sulphate, a glycosaminoglycan polysulphate, carrageenan or chondroitin sulphate E.

22. The method according to any one of Claims 17 to

21 wherein the monoclonal antibody is MAb^*5-B-10.

23. The method according to claim 22 wherein the labelled antibody is an anti-mouse antibody.

24. The method according to claim 23 wherein the labelled antibody is an anti-mouse IgM antibody.

25.The method according to any one of claims 17 to 24 wherein the enzyme is selected from an enzyme, a fluorescent label, a radiolabel and biotin.

26. The method according to claim 25 wherein the antibody is alkaline phosphatase conjugated rabbit anti-mouse IgM.

27. 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 substitution; and

■a labelled monoclonal antibody according to claim 6 or 7.

28. The kit according to claim 27 wherein the solid support is selected from PVC, polystyrene, agarose and sepharose.

29. The kit according to claim 28 wherein the solid support is PVC.

30. The kit according to any one of claims 27 to 29 wherein the polysulphated polysaccharide coating is selected from pentosan polysulphate, dextran sulphate, chondroitin sulphate and glycosaminoglycan polysulphates.

31. The kit according to claim 30 wherein the polysaccharide coating is pentosan polysulphate.

32. The kit according to any one of claims 27 to 31 wherein the labelled monoclonal antibody is MAb 5-B-10.

33. The kit according to claim 32 wherein the label is alkaline phosphatase.

34. 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 antibody according to any one of claims 1 to 5; and a labelled antibody which recognises the monoclonal antibody.

35. The kit according to claim 34 wherein the solid support is selected from PVC, polystyrene, agarose and sepharose.

36. The kit according to claim 35 wherein the solid support is PVC.

37. The kit according to any one of claims 34 to 36 wherein the polysulphated polysaccharide coating is selected from pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphates, carrageenan and chondroitin sulphate E.

38. The kit according to claim 37 wherein the polysulphated polysaccharide coating is pentosan polysulphate or dextran sulphate.

39. The kit according to any one of claims 34 to 38 wherein the monoclonal antibody is MAb 5-B-10.

40. The kit according to any one of claims 34 to 39 wherein the labelled antibody is selected from anti-mouse immunoglobulin antibodies.

41. The kit according to claim 40 wherein the labelled antibody is an alkaline phosphatase labelled rabbit anti-mouse IgM antibody.

42. The diagnostic kit of any one of claims 27 to 41 wherein the solid support is coated with poly-L-lysine.

43. The diagnostic kit according to claim 42 wherein the poly-L-lysine coated support is provided coated with the polysulphated polysaccharide.

44. A method for determining whether a polysulphated polysaccharide is a potential antiarthritic, anti-viral, anti-inflammatory, anti-coagulant or anti-degenerative drug which method comprises: determining whether the polysulphated polysaccharide is recognized by a monoclonal antibody according to any one of claims 1 to 5.

45. The method according to claim 44 wherein the monoclonal antibody is MAb 5-B-10.

46. 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 specimen, comprising administering a labelled antibody according to claim 6 or 7 to the tissue or specimen.