CA2005793A1 - Method for the detection and quantification of hyaluronan - Google Patents

Abstract

This invention provides a method for the detection and/or quantification of HA in biological samples. The method is an inhibition method using a hyaluronan binding protein and hyaluronan bound to a solid support.
The invention also provides a diagnostic kit for use in the method of the invention.

Description

-METHOD FOR THE Vr lr- 1 lON AND OUANTIFI~-~TTON OF ~AT.T~N~N
Technical Fiel~
The present invention relates to an improved method of detecting hyaluronan in biological fluids and to a diagnostic kit for use in that method.
Background Art Hyaluronic acid or hyaluronan (HA) is a linear unbranched polysaccharide consisting of repeating disaccharide units of B-(1,4)-D-glucuronic acid-B-(1,3)-D-N-acetylglucosamine. HA is widely distributed in vertebrate connective tissues, particularly umbilical cord, synovial fluid, vitreous aqueous humor, dermis, cartilage and intervertebral disc. In cartilage, it has been shown to be an important component for the macromolecular organisation of cartilage proteoglycan subunits. HA
provides the backbone for multiple site interactions with proteoglycan monomers, the interaction being electrostatic in nature and stabilized by link protein (Hascall et ~1-, 1974).
The importance of HA in maintaining the unique rheological properties of joint synovial fluid (SF) is well known (Balazs, 1970). Hyaluronic acid confers high viscosity and exclusion properties to SF, however its role in joint lubrication remains controversial even though recent evidence has been put forward to support its contribution to this important function (Langer Q~ al., 1988). In rheumatoid arthritis, the HA of joint synovial fluid is depolymerised and the concentration of HA in the CA~005793 blood of these patients is also elevated. Recently, it has been shown that serum levels of HA are also increased in a number of diseases including scleroderma (Engstrom-Laurent et al., 1985), and liver disease (Engstrom-Laurent et al., 1985; Frebourg ~ al., 1986). The quantification of HA in tissues and body fluids is thus emerging as a useful adjunct to diagnosis.
The quantification of microgram amounts of HA has been achieved by a variety of methods, the oldest and most common being by colorimetry using modifications of the carbazole method for the determination of hexuronic acid (Bitter ~ al., 1962). However, this method is not specific, since other hexuronate containing compounds and neutral sugars also generate colour in this assay. Some selectivity may be achieved using specific hyaluronidases (e.g. from Streptomyces hyal~rolyticus~ to generate reducing end groups which can then be quantified using thiobarbiturate (Rowley et al., 1982), however the sensitivity of this procedure is limited to a microgram or more of HA. In recent years, methods employing proteins with a specific affinity for HA have been utilised for its estimation. Tengblad (Tengblad, 1979; Laurent et al., 1980) developed a radiometric assay for HA using HA-binding proteins (HABP) isolated from bovine cartilage. This method has enabled submicrogram quantities of HA to be detected and quantified in biological samples, but since 5I-HABP has a relatively short half-life, the assay must be performed with freshly prepared material. An ELISA method has been C~2005793 devised by Delpech and co-workers (Delpech ~ ~1., 1985), using the HA binding protein, hyaluronectin. However the method requires pre-coating of the plates with another HA
binding protein and this has presented some difficulties in the routine application of this approach.
European Patent Application No. 87850360.6 (Publication No. 0 271 461) describes a method of detecting HA by an inhibition technique using a derivatized HA and an HA binding structure. The specification recites that the reaction between the HA binding structure and HA is sensitive to pH variations and excessive temperatures. Thus it is a feature of the assay described therein that the assay is conducted at a pH within the range of 5.8 to 7.3 and at a temperature in the range of 0C to 27C.
The present inventors consider that HABP would not assume native conformation in that pH range and that consequently a more alkaline pH is desirable to ensure that disulphide bridges present in the native form of HABP are maintained. This is supported in the literature [Plaas et al, 1986]
European Patent Application No. 87850360.6 describes the direct binding of HA to a solid support. This has also been described by other workers (Delpech et al, 1985).
The present inventors have found that in their hands HA does not bind well to solid support materials. To improve the binding of HA to the solid support materials, the present inventors have employed a polycationic material interposed between the solid support material and the HA.

~ 5 ~ CA~005 7~3 Suprisingly, the present inventors have found that the inclusion of a polycationic coating material provides a highly sensitive assay for HA.
Descript;o~ of ~hP InV~nt;on Throughout the specification and claims, the term hyaluronan (HA) is intended to encompass hyaluronan and hyaluronic acid.
The present invention provides an improved method of detecting and/or quantifying hyaluronan in biological fluids and tissues which involves the interaction of hyaluronan in a sample with an excess of a labelled hyaluronan binding protein (HABP).
The method was developed by modification of existing techniques (Tengblad, 1979; Laurent and Tengblad, 1980;
Delpech et al, 1985; Kongtawelert and Ghosh, 1989) and by utilization of the specific and strong binding of streptavidin to biotin containing molecules (Wilchek and Bayer, 1984a; 1984b). HA is an anionic macromolecule which should not bind directly to polyvinyl chloride or polystyrene plates. Immobilization of this macromolecule to wells of microtitration plates has previously been described using cationic proteins such as protamine (Keiser, 1987), however, these proteins can introduce non-specific interactions which can interfere with the determination of HA. This problem was overcome in the present method by using high molecular weight poly-L-lysine HCl pre-coated plates to immobilize the HA. Significant improvement of the binding of HA on the pre-coated plate which saturated when the concentration of poly-L-lysine was over 50 ~g/ml has been demonstrated by the present inventors. A similar technique was reported by Epstein and Lunney (Epstein and Lunney, 1985) for the development of an ELISA for cell surface antigens.
According to a first embodiment of the present invention there is provided a method for the detection and/or quantification of hyaluronan in a sample containing HA which method comprises:
providing a solid support coated with a polycationic coating substance, the polycationic coating substance coated support being further coated with a source of HA;
exposing the sample to a labelled HABP;
applying the labelled HABP containing HA containing sample to the support;
removing unbound sample; and detecting the labelled HABP, wherein the method is carried out at an alkaline pH.
Preferably the method comprises:
providing a solid support;
coating the solid support with the polycationic coating substance; and coating the coated support with the source of HA.
In an alternative preferred form the method comprises;
providing a solid support coated with the polycationic coating substance;
and coating the coated support with the source of HA.

I~ A 20057 q;~

Typically, the polycationic coating substance is selected from poly-L-lysine, polyarginine, glutaraldehyde and a cationic protein, which does not produce non-specific interactions.
Preferably the polycationic coating substance is poly-L-lysine.
Typically, the cationic protein is secretory proteinase leukocyte inhibitor.
Preferably the method is carried out at a pH between 7.5 and 9Ø
More preferably the method is carried out at a pH of 8.6.
Typically, the method is carried out at a temperature between 18C and 37C.
Preferably the method is carried out at 37C.
Typically, the solid support is selected from polystyrene, PVC, Sepharose and agarose.
Preferably the support is PVC.
The support may be formed as a microtitre plate, tube or bead.
Typically the HA coating source is derived from synovial fluid, cocks comb, umbilical cord, serum, plasma dermis, cartilage, intervertebral disc or a microorganism.
Preferably the HA coating substance is derived from umbilical cord.
Typically the HABP is derived from a proteoglycan monomer.

~,A2005793 Typically the proteoglycan monomer is derived from animal hyaline cartilage.
Preferably the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.
The HABP may be labelled with an enzyme, a radioisotope, a fluorescent label or biotin.
Typically the label is: an enzyme selected from alkaline phosphatase, horse radish peroxidase, ~-galactosidase and urease; a radioisotope selected from 125I and 131I; a fluorescent label selected from a fluorochrome, FITC and TRITC or biotin.
Preferably the label is biotin.
More preferably the biotinylated HABP is detected using a labelled avidin or streptavidin.
More preferably the labelled avidin or streptavidin is enzyme labelled.
More preferably the avidin or streptavidin is labelled with an enzyme selected from alkaline phosphatase, horse radish peroxidase, ~-galactosidase and urease.
Most preferably the avidin or streptavidin is labelled with alkaline phosphatase.
Where enzymes are used for labelling either a single enzyme, an oligomeric form of the enzyme, or an enzyme/antienzyme complex may be used.
Further, the enzyme may be coupled to an alternative detection system, such as an amplification system.
For instance, the signal from alkaline phosphatase can be amplified 100-5000 fold using a redox cycle based on - g - ~JA2005793 the cycling of NAD.
Enzyme amplification methods are described in Bates (1987).
Typically the sample is a synovial fluid sample, vitreous aqueous humor, dermis, cartilage, serum, plasma or intervertebral disc.
According to a second embodiment of this invention there is provided a diagnostic kit for use in the detection and/or quantification of hyaluronan in a sample containing hyaluronan the kit comprising:
a solid support;
a polycationic coating material;
an HA coating material;
a labelled HABP; and an HA standard.
Preferably, the kit is provided with the solid support coated with the polycationic material and the HA
coating coated on the coated solid support.
In an alternative preferred form the solid support is provided coated with the polycationic coating material and the HA coating is provided to be applied to the coated support.
Typically the solid support is PVC, polystyrene, agarose or Sepharose.
Preferably, the solid support is PVC.
The support may be formed as a microtitre plate, tube or bead.

- 10 _ ~ A~005 193 Typically the polycationic coating material is selected from poly-L-lysine, polyarginine, glutaraldehyde and a cationic protein, which does not produce non-specific interactions.
Typically, the cationic protein is secretory proteinase leukocyte inhibitor.
Preferably the polycationic material is poly-L-lysine.
Typically the HA coating source is derived from synovial fluid, cocks comb, umbilical cord, serum, plasma, dermis, cartilage, invertebral disc, or a microorganism.
Preferably, the HA coating substance is derived from umbilical cord.
Typically the HABP iS derived from a proteoglycan monomer.
Typically the proteoglycan monomer is derived from animal hyaline cartilage.
Preferably the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.
Typically the HABP iS labelled with an enzyme, a radioisotope, a fluorescent label or biotin.
Typically, the enzyme is selected from alkaline phosphatase, horse radish peroxidase, ~-galactosidase and urease.
Typically, the radioisotope is selected from 125I
and 131I
Typically, the fluorescent label is a fluorochrome, FITC or TRITC.
Preferably the label is biotin.

~2005 79~

-Typically, the biotinylated HABP is detected using a labelled avidin or streptavidin.
Preferably, the avidin or streptavidin is enzyme labelled.
More preferably the label is selected from alkaline phosphatase, horse radish peroxidase, ~-galactosidase and urease.
Most preferably the label is alkaline phosphatase.
Typically the HA standard is selected from Healon R
and human umbilical cord HA.
Where enzymes are used for labelling either a single enzyme, an oligomeric form of the enzyme, or an enzyme/antienzyme complex may be used.
Further, the enzyme may be coupled to an alternative detection system, such as an amplification system.
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 th~ Draw;n~s - 20 Figure 1 is a diagrammatic representation of the principle used for the quantification of HA by the labelled avidin - biotin technique.
Figure 2 shows a typical standard inhibition curve obtained when quantifiying HA by the labelled avidin-biotin technique using HealonR in 6% BSA as a standard reagent.
Figure 3 shows an elution profile of HABP on HA-Sepharose affinity chromatography. The arrow indicates the starting point for elution with 4M GuHCl.

~ ~2005 793 Figure 4 shows a saturation curve obtained for the optimal concentration of human umbilical cord HA in PBS, pH
7.4 for absorption on poly-L-lysine (50~/ml; 100~g/well) pre-coated polyvinyl chloride immunoassay plates.
Figure 5 shows the relationship between dilution of HABP and alkaline phosphatase conjugated streptavidin at dilutions of 1:1000, 1:2000 and 1:4000 on an HA coated plate.
Figure 6 shows a comparison of the sensitivity of the assay of the invention with the Pharmacia HA test and the ELISIA assay.
Figure 7 shows a comparison of amounts of poly-L-lysine for precoating plates.
R~T METHOD OF C~RPYING OUT THE INVENTION
The samples in which the HA is to be detected are prepared in accordance with standard techniques of sample preparation.
The preparation of coated plates, tubes or beads may be carried out in accordance with standard techniques.
The HABP for use in the method of the invention may be derived from a variety of sources and is prepared in accordance with standard techniques.
The HA coating and standard materials may be derived from a variety of sources and are isolated and formulated for use in accordance with standard techniques.
Standard curves for the estimation of HA in unknown samples was in accordance with standard techniques for the preparation of standard curves.

c A20057q3 The preparation of the diagnostic kits of the invention is also in accordance with standard techniques.
Esample 1: Detection ~n~ quantification of ~A ~y measurinq ~s h;otinylat~_~ARP
A. Isolation of HABP
Proteoglycans (PGs) were extracted from human articular cartilage with buffered 4M guanidine hydrochloride, pH7.4 containing protease inhibitors and purified under associative and dissociative conditions by ultracentrifugation, as described previously by Heingard (Heingard et al, 1977). The purified PGs were digested with chondroitinase ABC to remove chondroitin sulphate chains and the products separated by Sepharose CL-2B column chromatography (Caterson et al., 1983). The keratan sulphate core protein fraction was further partially digested with trypsin. The HABP can be obtained by purification further using HA-affinity chromatography (Tengblad, 1979) and the bound peak which eluted with 4M
GuHCl was then dialysed against distilled water, lyophilised and stored frozen until required. Lyophilised HABP were further purified by Sepharose CL-6B (1.5 x 3.0cm) and Sephacryl HR S-300 column chromatography (3 x lOOcm) using 4M GuHCl as eluent buffer and running at a flow rate of 0.25ml/min at 4C. HABP was collected, dialyzed against distilled water and then lyophilized.
B. B;otinylation of ~BP
Biotinylation of HABP was performed by the method of Rappuoli ç~ al, (1981). HABP prepared as in A above, was - 14 _ C A2005793 dissolved in 0.lM sodium hydrogen carbonate buffer pH 8.5 and mixed in a ratio of 1:3 (w/w) with N-hydroxysuccinimidobiotin (34.lmg/ml of dimethylsulfoxide) at room temperature for 18 hours. The mixture was applied to a Sephadex G-25 column and eluted with 0.lM Tris-0.05M
NaOAc pH 8.6. The excluded protein peak was collected, aliquoted and stored at -20C as stock solution.
C. Coating Microtitratio~ Plates wi~h ~
Activated polyvinyl chloride immuno-assay plates were pre-coated with poly-L-lysine hydrochloride by the addition of 100~1/well of a 50~g/ml solution in distilled water.
After incubation for 1 hour at 37C, the solution were flicked out and the plates air-dried. To the pre-coated plates was added 100~1/well of human umbilical cord HA
(50~g/ml in PBS pH 7.4). The plates were then incubated at 37C for 1 hour, washed three times with PBS-Tween 20 (0.05%
v/v) and 1% BSA-PBS pH 7.4 (100~1/well) was added. A
further incubation at 37C for 1 hour followed by 3 times washing with PBS-Tween 20 (0.05% v/v) and air-drying afforded the required HA-plates. The coated HA-plates were wrapped in polythene film and stored at 4C until needed.
D. The guantification of HA in sa~plPc ~y ~hP l~helled avi~lin-hiotin tPrhni~lle (~ r.~B TP-~hniqlle) The principle of this method is shown in Figure 1.
Samples containing unknown amounts of HA or standard of known concentrations (between 6-200pg/ml) of a highly purified HA preparation (Healon ) in PBS pH 7.4 containing 6% BSA were pipetted into small polypropylene tubes with ~ A2005 793 appropriate concentrations of biotinylated-HABP [B-HABP]
(equal volume of 175~1 each) in 0.lM Tris-0.lM NaOAc pH
8.6, mixed using a vortex mixer and incubated at 37C in a waterbath for 3 hours. Aliquots (100~1) of this reaction mixture (in triplicate for each sample) were then applied to HA-coated and BSA-blocked plates and incubated at 37C for 90 minutes. The plate wells were washed 5 times with PBS-Tween 20 (0.05% v/v) and the appropriate dilution of alkaline phosphatase conjugated streptavidin (100~1/well;
Calbiochem, USA; Cat. No. 189732) was added. The plates were then washed 5 times with PBS-Tween 20 (0.05% v/v) and air-dried. Alkaline phosphatase substrate [lmg/ml of p-nitrophenyl phosphate in lM diethanolamide (Ajax Chemical, Australia; Cat. No. 1019) and 0.001M MgC12, pH 9.8;
100~1/well] was added and plates incubated at 37C for 60 minutes. The reaction was stopped by the addition of 50~1 of 5N NaOH. The absorbance at 405/690nm was determined using a microtiter plate reader (Titertek Twinreader, Flow Laboratories, Zwanenburg, The Netherlands).
For this assay, triplicate results were averaged and the precentage inhibition was calculated by the following formula:
[A405(B-HABP+sample)-A405(BSA)]xl00 % Inhibition = 100 - ----------------------------------[A405(B-HABP+BSA)-A405(BSA)]

A standard inhibition curve for HA was constructed using log/linear co-ordinates and the HA levels in the test CA200~793 samples were determined by comparing their capacity to inhibit colour development at OD 405/690nm relative to this standard curve.
Figure 2 shows a typical standard inhibition curve for HA using the standard conditions described. It was found using this method that it was possible to reproducibly quantify between 10-200pg/ml of HA in biological samples.
Figure 6 illustrates the improvement in sensitivity by this method compared with ELISIA assay and the Pharmacia HA test.
In sera taken from healthy volunteers the intra- and inter-assay variation coefficients obtained using this assay were 14 + 6% and 19 + 9% respectively. The HA was determined by adding know amounts of purified HA solutions (HealonR) to samples. The recovery obtained varied between 75 and 98% of the theoretical value.
The mean value of HA in diluted normal sera was 24 +
12ng/ml by our labelled-avidin biotin technique and 22 +
16ng/ml using a commercial radioassay kit (HA-test, Pharmacia, Sweden; see Table 1). Using the same sera samples a correlation coefficient of 0.785 was obtained using values generated using our assay and the commercial kit. The value for HA obtained by the two independent methods were found not to be significantly different from each other using Student paired T-test (Table 1).
E~ample 2: Detectio~ of ~A in ar~hriti~ ~t;~nt~
The values determined for the human sera and synovial fluids taken from RA and OA patients, are shown in Table 2.

- 17 - ~ A200~ 7~3 The concentration of HA in sera obtained from RA patients was, in the small number of samples examined, higher than in sera from OA patient (Table 2). However, for sera from both RA and OA patients the HA levels were statistically (p < 0.0001) higher than in sera of healthy volunteers (Table 2). There did not appear to be any difference between the level of HA in synovial fluid derived from RA
and OA joints.
E~mple 3: Optimisation of the ~say An important reagent for this assay was the HA-binding protein which was prepared as described herein or according to methods reported by others (Bonnet ~ al, 1981;
Tengblad, 1979). Figure 3 shows the elution profile obtained by HA-affinity column chromatography of the trypsin digested PG-monomers. As is evident, HABP was eluted from the column by 4M GuHCl. In order to determine capacity of the poly-L-lysine pre-coated plates for HA they were incubated with various concentrations of human umbilical cord HA. The amount of HA adhering to the poly-L-lysine pre-coated plates was determined by adding B-HABP, enzyme-conjugated streptavidin and enzyme substrate, as described above. The saturation curve for HA using poly-L-lysine pre-coated plates is shown in Figure 4. It was found that the plates were saturated at concentrations of HA above 50~g/ml. This concentration of HA was therefore used in the standard method which had a low coefficient of variation from well to well (data not shown).

CA2005 1~3 To determine the optimal dilution of B-HABP and enzyme conjugated streptavidin, various dilutions of B-HABP
were applied to HA-coated plates and incubated with different dilutions of enzyme conjugated streptavidin.
Figure 5 shows the titration curves obtained. A dilution of B-HABP of 1:40 was selected together with an enzyme conjugated streptavidin dilution of 1:2,000 as observed by the absorption at OD 405nm using a development time of 60 minutes at 37C. Under these conditions optimum quantification of HA in standards and unknown samples was obtained.
The conditions established for this assay only employed very small amounts of B-HABP (approximately 40~g per 32 samples of one assay plate) which is a distinct advantage as native HABP is relatively difficult to prepare in large quantities. In contrast to other assays (Tengblad, 1979; Laurent and Tengblad, 1980), we used HABP derived from human articular cartilage, however, we have found that the bovine HABP is just as effective. Under the standard conditions of the assay it was found that serum samples required 1000-time dilution in PBS pH 7.4 and 6% BSA to enable HA levels to be accurately determined. The high dilution of serum samples required in this present assay eliminated the need to precipitate proteins from samples with 6% trichloroacetic acid, followed by dialysis and lyophilization prior to analysis as is required in other assays. This method also avoids the problems associated with the presence of naturally occurring antibodies against Ch2l)05793 HA in biological fluids of some species (Underhill, 1982) as well as some proteins e.g. haptoglobin, inter-a-trypsin inhibitor and al-proteinase inhibitor in biological fluids (Hutadilok et al, 1987). These antibodies and other proteins (at physiological concentrations) may interfere with immunologically based assays (Delpech et al, 1985) giving an underestimation of HA.
The major advantage of the present method against other published HA assays is its ability to allow HA to be conveniently determined in extremely small volumes of biological fluids. For example, experiments have shown that 10~1 of sera (equivalent to that obtained from a needle finger bleeding) provides a sufficient amount for reproducible analysis of HA.
The level of HA is reported to be elevated in rheumatoid arthritis, neoplastic diseases, and liver diseases (Engstrom-Laurent et al, 1985a; Frebourg et al, 1986; Engstrom-Laurent et al, 1985b; Engstrom-Laurent and Hallgren, 1985). Using the present assay it was possible to confirm previous reports (Engstrom-Laurent and Hallgren, 1985) that sera of patients with RA contained higher levels of HA than normals or OA patients (Table 1 and 2). While the reasons for this have yet to be clarified, the increased mobilization of HA into the blood from the synovial space may be related to inflammation which enhances the permeability of the synovial membrane (Harris, 1981), as well as increasing interstitial lymphatic drainage of HA
(Label et al, 1989) in arthritis and even some physical exercise. Certainly there appears to be some correlation between joint inflammation and circulating HA levels as determined by experimental studies in animals (Goldberg et al, 1988; Bjork et al, 1989).
Irrespective of the explanation, it is clear that the measurement of the level of HA in biological fluids provides a useful marker for monitoring the onset and progression of a number of important disease and disorders. This being the case, the availability of a simple sensitive and accurate assay for this glycosaminoglycan can only improve our understanding and treatment of these diseases. The labelled avidin-biotin technique for HA which has been described here would appear to fulfill this criterion.
Indust~ial P~plications The present invention is of use in the detection of hyaluronan in biological fluids and tissues. The detection of hyaluronan is a useful adjunct to the diagnosis of diseases such as rheumatoid arthritis, neoplastic diseases, scleroderma and liver disease.

- 21 - Cd2~05 7q3 ~ :N~.~
Balazs, E.A., and Gibbs, D.A. The rheological properties and biological function of hyaluronic acid. In: Chemistry and molecular biology of the intercellular matrix (Balazs, E.A., ed.), vol. 3, pp.l241-1253, 1970, Academic Press, New York.
Bates, D.L., Enzyme amplification in diagnostics, Tibtech 5:
204-209, 1987.
Bitter, T. and Muir, H.M. A modified uronic acid carbazole reaction. Anal. Biochem., 4: 330-334, 1962.
Bjork, J., Kleinau, S., Tengblad, A., Smedegard, G. Elevated levels of serum hyaluronate and correlation with disease-activity in experimental-models of arthritis.
Arthritis Rheum., 32(3): 306-311,1989.
Bonnet F, Bunham DG, and Hardingham TE (1985): Structure and interactions of cartilage proteoglycan binding region and link protein. Biochem J 228 77-85.
Caterson, B., Christner, J.E., Baker, J.R. Identification of monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate - monoclonal antibodies to cartilage proteoglycan. J. Biol. Chem., 258 (14): 8848-8854, 1983.
Delpech, B., Bertrand, P. and Mainconnat, C.
Immunoenzymoassay of the hyaluronic acid -hyaluronectin interaction: application to the detection of hyaluronic acid in the serum of normal subjects and cancer patients. Anal. Biochem., 149 : 555-565, 1985.
Engstrom-Laurent, A. and Hallgren, R. Circulating - 22 - C A20 05 7~

hyaluronate in rheumatoid arthritis: relationship to inflammatory activity and the effect of corticosteroid therapy. Ann. Rheum. Dis., 44: 83-88, 1985.
Engstrom-Laurent, A., Felteliu., N., Hallgren, R. and Wasteson, A. Raised serum hyaluronate levels in scleroderma - an effect of growth-factor induced activation of connective-tissue cells. Ann. Rheum.
Dis., 44: 614-620, 1985a.
Engstrom-Laurent, A., Loof, L., Nyberg, A. and Schroder, T. Increased serum levels of hyaluronate in liver disease. Hepatology, 54: 638-642, 1985b.
Epstein, S.L. and Lunney, J.K. A cell surface ELISA in the mouse using only poly-L-lysine as cell fixative. J.
Immunol. Methods, 76: 63-72, 1985.
Frebourg, T., Delpech, B., Bercoff, E., Senant, J., Bertrand, P., Deugnier, Y. and Bourreille, J. Serum hyaluronate in liver diseases - study by enzymoimmunological assay. Hepatology, 6: 392-395, 1986.
0 Goldberg, R.L., Lenz, M.E. and Thonar, E.J.-M.A. Levels of plasma hyaluronate in osteoarthritis and rheumatoid arthritis. Trans. Orthop. Res. Soc., 491: 1988.
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- 23 - C A~05 793 Chem., 249: 4242-4249, 1974.
Heinegard, D. Polydispersity of cartilage proteoglycans:
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Hutadilok, H., Ghosh, P., and Brooks, P.M. Binding of haptoglobin, inter-a-trypsin inhibitor, and al- proteinase inhibitor to synovial fluid hyaluronate and the influence of these proteins on its degradation by oxygen derived free radicals. Ann.
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CA20057~3 .

TABLE

The Levels of HA in Sera of Normal Healthy Volunteers as Determined by a Commercial Radioassay (HA-test, Pharmacia) and the Labelled Avidin-Biotin Technique Hyaluronan (ng1ml) Subjects Sex Age (ye~
HA-Test Labelled Avidin-Biotin Test Mean + S.D. 22T 16 23+11 ~l b ~ A2U05 7~33 ~ TABL~: 2 The HA Levels in Pathological Human Sera and Synovial Fiuids as Determined by Labelled Avivin-Biotin Test Hyaluronan by HA-LAB Test Subjects Diagnosis -Serum (ng/ml) Synovial Fluid (llglml) JN RA 150 *N.D.

JM RA *N.D. 255 Mean + S.D. 91+ 39 363 + 154 *N.D. = Not determined

Claims (55)

1. A method for the detection and/or quantification of hyaluronan in a sample which method comprises:
providing a solid support coated with a polycationic coating substance, the polycationic substance being further coated with a source of HA;
exposing the sample to a labelled HABP;
applying the labelled HABP containing sample to the support;
removing unbound sample; and detecting the labelled HABP, wherein the method is carried out at an alkaline pH.

2. The method according to claim 1 comprising:
providing a solid support;
coating the support with a polycationic coating substance; and coating the coated support with a source of HA.

3. The method according to claim 1 or claim 2 comprsing:
providing a solid support coated with a polycationic coating substance; and coating the coated support with a source of HA.

4. The method according to any one of claims 1 to 3, wherein the polycationic coating substance is selected from poly-L-lysine, poly-arginine, glutaraldehyde and a cationic protein which does not produce non-specific interactions.

5. The method according to claim 4 wherein the cationic protein is secretory-proteinase leukocyte inhibitor.

6. The method according to any one of claims 1 to 5 wherein the assay is carried out at a pH between 7.5 and 9Ø

7. The method according to claim 6 which is carried out at a pH of 8.6.

8. The method according to any one of claims 1 to 7 which is carried out at a temperature between 18°C and 37°C.

9. The method according to claim 8 which is carried out at 37°C.

10. The method according to any one of claims 1 to 9 wherein the solid support is selected from polystyrene, PVC, agarose and Sepharose.

11. The method according to claim 10 wherein the solid support is PVC.

12. The method according to any one of claims 1 to 11 where the support is a microtitre plate, a tube or a bead.

13. The method according to any one of the claims 1 to 12 wherein the source of HA for coating is derived from synovial fluid, cockscomb, umbilical cord, serum, plasma, dermis, cartilage, intervertebral disc or a microorganism.

14. The method according to claim 13 wherein the HA
coating substance is derived from umbilical cord.

15. The method according to any one of claims 1 to 14 wherein the HABP is derived from a proteoglycan monomer.

16. The method according to claim 15 wherein the proteoglycan monomer is derived from animal hyaline cartilage.

17. The method according to claim 16 wherein the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.

18. The method according to any one of claims 1 to 17 wherein the label is an enzyme label.

19. The method according to claim 18 wherein the enzyme is selected from alkaline phosphatase, horse radish peroxidase, .beta.-galactosidase, and urease.

20. The method according to any one of claims 1 to 17 wherein the label is a radioactive label.

21. The method according to claim 20 wherein the radioactive label is selected from 125I, and 131I.

22. The method according to any one of claims 1 to 17 wherein the label is a fluorescent label.

23. The method according to claim 22 wherein the fluorescent label is selected from a fluorochrome, FITC and TRITC.

24. The method according to any one of claims 1 to 17 wherein the label is biotin.

25. The method according to claim 24 wherein the biotinylated HABP is detected using a labelled avidin or streptavidin.

26. The method according to claim 25 wherein the labelled avidin or streptavidin is enzyme labelled.

27. The method according to claim 26 wherein the enzyme label is selected from alkaline phosphatase, horse radish peroxidase .beta.-galactosidase and urease.

28. The method according to claim 27 wherein the enzyme label is alkaline phosphatase.

29. The method according to claim 18 or claim 26 wherein the enzyme label comprises an amplified enzyme label.

30. The method according to any one of claims 1 to 29 wherein the sample is a synovial fluid sample, vitreous aqueous humor, dermis, cartilage, serum, plasma, intervertebrate disc or a microorganism.

31. A diagnostic kit for use in the detection and/or quantification of hyaluronan in a sample containing hyaluronan the kit comprising:
a solid support;
a polycationic coating material;
an HA coating;
a labelled HABP and an HA standard.

32. The diagnostic kit according to claim 31 wherein the solid support is coated with the polycationic material.

33. The diagnostic kit according to claims 31 or 32 wherein the solid support is coated with the polycationic material and the polycationic material is coated with the HA
coating.

34. The diagnostic kit according to any one of claims 31 to 33 wherein the solid support is selected from PVC, polystyrene, agarose and Sepharose.

35. The diagnostic kit according to claim 34 wherein the solid support is PVC.

36. The diagnostic kit according to any one of claims 31 to 35 wherein the solid support is in the form of a microtitre plate, a tube or a bead.

37. The diagnostic kit according to any one of claims 31 to 36 wherein the polycationic coating material is selected from poly-L-lysine polyarginine, glutaraldehyde and a cationic protein which does not produce non-specific interactions.

38. The diagnostic kit according to claim 37 wherein the cationic protein is secretory proteinase leukocyte inhibitor.

39. The diagnostic kit according to any one of claims 31 to 38 wherein the HABP is derived from a proteoglycan monomer.

40. The diagnostic kit according to claim 39 wherein the proteoglycan monomer is derived from animal hyaline cartilage.

41. The diagnostic kit according to claim 40 wherein the animal hyaline cartilage is articular cartilage from a human, bovine, porcine or lapine.

42. The diagnostic kit according to any one of claims 31 to 41 wherein the source of HA for coating is derived from synovial fluid, cockscomb, umbilical cord, serum, plasma, dermis, cartilage, intervertebral disc or a microorganism.

43. The diagnostic kit according to claim 42 wherein the HA coating substance is derived from umbilical cord.

44. The diagnostic kit according to any one of claims 31 to 43 wherein the HABP is labelled with an enzyme.

45. The diagnostic kit according to claim 44 wherein the enzyme is selected from alkaline phosphatase, horse radish peroxidase, .beta.-galactosidase and urease.

46. The diagnostic kit according to any one of claims 31 to 41 wherein the HABP is labelled with a radioactive isotope.

47. The diagnostic kit according to claim 46 wherein the radioactive isotope is selected from 125I and 131I.

48. The diagnostic kit according to any one of claims 31 to 41 wherein the label is a fluroescent label.

49. The diagnostic kit according to claim 48 wherein the fluorescent label is selected from a fluorochrome, FITC
and TRITC.

50. The diagnostic kit according to any one of claims 31 to 41 wherein the label is biotin.

51. The diagnostic kit according to claim 50 where the biotinylated HABP is detected with labelled avidin or streptavidin.

52. The diagnostic kit according to claim 51 where the labelled avidin or streptavidin is enzyme labelled.

53. The diagnostic kit according to claim 52 where the enzyme label is selected from alkaline phosphatase, horse radish, peroxidase, .beta.-galactosidase and urease.

54. The diagnostic kit according to claim 53 where the enzyme label is alkaline phosphatasee.

55. The diagnostic kit according to claim 44 or claim 52 where the enzyme label comprises an amplified enzyme label.