JP2003502054A - Heparanase assay - Google Patents

Abstract

(57) Summary (i) A sample is incubated in the presence of a first solid support having immobilized HSGAG polymer substrate for heparanase thereon, said substrate being insensitive to the action of proteases Wherein the substrate has a first binding moiety attached thereto, and further has a paracrine cell regulator capable of binding to the HSGAG bound thereto, (ii) incubating the incubated sample with an HSGAG polymer substrate and the second moiety. A second solid support having a second portion provided thereon, which can be cleaved from the first solid support to the second support by binding to the paracrine cell regulator or the first binding portion. Incubating and (iii) generating a measurable signal on the other of the first or second portion of the cleavage substrate immobilized on the second support solid phase; Comprises measuring the signals on the second solid support is separated from the lifting body, assay of a sample for measuring heparanase activity.

Description

Detailed Description of the Invention

[0001]   The present invention relates to assays for heparanase activity.

Endoglycosidases are capable of cleaving polymers composed of glycosaminoglycan units (so-called “GAG polymers”). Enzymes that cleave GAG polymers are known per se as endoglycosidases and include heparanase, which cleaves heparan sulfate GAG, hyaluronidase, which cleaves hyaluronate GAG, and chondroitinase, which cleaves chondroitin sulfate GAG.

Heparanase has been described in T and B lymphocytes, monocytes, neutrophils, platelets, endothelial cells and liver and pancreatic tissues. This endoglycosidase activity that degrades heparan sulphate GAG is variously referred to as heparanase, heparinase, heparitinase and heparan sulphate lyase, and most families of enzymes with different properties with respect to the initial cleavage site in GAG polymers. Definitely show.

An example of an anticoagulant, heparin, is heparan sulfate glycosaminoglycan (H
SGAGS) is a highly N- or O-sulfated glucuronic (in some cases iduronic) acid glucosaminase (in some cases N-acetylated) carbohydrate polymer. The high anionic charges are not randomly distributed in the polymer, but are believed to form clusters of charged residues that confer specific functional binding properties on the sequence.

HSGAG is present in the extracellular matrix, in the basement membrane and on the cell surface.
HSGAG is known to play important roles in cross-linking basement membrane component structural proteins, controlling membrane permeability (best explained in glomerular basement membrane) and controlling cytokine and growth factor bioavailability.

Heparanase is known to play an important role in the following areas of drug biology: a) Cancer metastasis; most metastatic cells show the highest level of heparanase enzyme, which is a cancer cell. But in order to promote tissue invasion, HSGAG in ECM and basement membrane
Can be decomposed. Cancer cells that secrete heparanase have angiogenic,
It may support tumor angiogenesis and thus tumor growth. This is a major area for the development of novel antagonists to heparanase as part of a therapeutic strategy to suppress and inhibit metastasis.

B) Inflammatory joint diseases such as rheumatoid arthritis; angiogenesis in joints can support chronic inflammatory lesions. Agents that inhibit heparanase in joints may reduce angiogenesis, thus inhibiting the formation of tissue inflammatory pannus leading to cartilage degradation and bone erosion.

C) Proteinuria disease; HSGAG expression in the glomerular basement membrane regulates the passage of anionically charged proteins such as albumin. There is evidence that reduced basement membrane HSGAG is associated with microalbuminuria in early diabetic nephropathy. In addition, many clinical and experimental studies have demonstrated a reduction in proteinuria with low molecular weight heparin treatment. These findings support a role for increased heparinase expression in the glomerulus, which should be treated with appropriate antagonists.

The known assays for heparinase activity (summarized below) have many drawbacks. (i) In vitro metabolic radiolabeling of cell cultures with ³⁵ SO ₄ or ³ H incorporated into the radioactive substrate HSGAG is commonly used for the detection of heparanase. Radioactive label G
The AG may be used for subsequent isolation and purification by binding to a solid support. Alternatively, radiolabeled ECM seeded in culture dishes with the removed cell layer can be used as a substrate. Alternatively, purified HSGAG or heparin ³ H or ¹²⁵ I
Labeling is performed and the labeled substrate is bound to the solid phase. Heparanase activity is detected by the loss of radiolabel or a reduction in the molecular size of the labeled species.

Radioactive labeling is dangerous and requires special disposal methods. Large variations in substrate specific activity occur between batches. A batch of ³⁵ SO ₄ substrate has a short life of 2-3 months.

(Ii) SH substrates are sensitive to other enzymes. Heparan sulfate proteoglycans are used as substrates to release the labeled HSGAG or reduce the size characteristics of HSGAG (using isotopes or other labels) by molecular sieving. The assay, which is said to be a measurement of heparanase that detects apparent enzymatic activity by identification, is not specific to heparanase. A wide range of proteases present in the sample act on the proteoglycan component, solubilizing a significant amount of HS chains, resulting in size reduction by further removal of bound protein. Other enzymes, such as hyaluronidase, can cleave the substrate. Few current assays have proven unresponsive to this range of enzymes.

(Iii) Sensitivity and quantification Assays that require long incubation times (4-24 hours) to achieve show a lack of sensitivity, indicating the possibility of interference by non-specific factors (eg proteases). It remains. Assays that require molecular sieves to identify the consequences of heparanase activity are qualitative in nature.

(Iv) Multi-Step Handling of Samples Assays that require centrifugation or molecular sieving to separate products of heparanase activity can only be performed in small batch assays.

[0014]   It is an object of the present invention to avoid or reduce the above drawbacks.

In a first aspect of the invention, (i) the sample is incubated in the presence of a first solid phase support having immobilized HSGAG polymer substrate for heparanase thereon. The substrate is insensitive to the action of a protease, the substrate having a first binding moiety attached to it, and further having a paracrine cell regulator attached to it, which is capable of binding the HSGAG, (ii) the incubated sample A second having a second portion provided above
Incubation with a solid support where the HSGAG polymer substrate can be cleaved from the first solid support to the second support by attaching the second moiety to a paracrine cell regulator or a first binding moiety. (Iii) a measurable signal is generated on the other of the first or second portions of the cleaved substrate immobilized on the second supporting solid phase, and (iv) the second solid phase separated from the first solid phase support. Provided is a sample assay method for measuring heparanase activity, which comprises measuring a signal on a phase support.

In a second aspect, the present invention provides a solid support having an HSGAG polymer substrate for heparanase immobilized thereon for use in an assay as defined above (first support above). Same as the body), wherein the substrate has a first binding moiety bound thereon and further has a paracrine cell regulator capable of binding HSGAG bound thereto, wherein the first moiety or the paracrine The cell regulator can be associated with the second part.

In a third aspect, the invention is capable of binding to and forming a complex with a first solid support as defined in the preceding paragraph and a first portion or paracrine cell regulator provided thereon. A combination of a second solid support having a second portion is provided.

In a fourth aspect, there is provided an assay kit comprising the combination of the preceding paragraphs and a signal generating agent for generating a signal indicative of the result of the assay.

The paracrine cell regulator can be, for example, a growth factor, cytokine or chemokine.

The assay of the present invention allows for the rapid quantification of heparanase activity in a sample having, or possibly containing, such activity.

Although the present invention is further described with reference to the HSGAG-binding growth factor paracrine cell regulator, it should be understood that the invention is equally applicable to other paracrine cell regulators.

The assay is performed using the solid phase form. Assay step (i) uses a first solid phase support onto which is immobilized an HSGAG polymer substrate cleavable by heparanase present (or possibly present) in the sample. . The bond to the polymeric substrate is (a) HSGAG-bound growth factor, and (b) the first binding moiety.

The support-bound HSGAG substrate is insensitive to the action of proteases at the concentrations normally found in clinical samples (otherwise the assay gives false positive results). This can be accomplished by ensuring that the HSGAG substrate binds to the first solid support in a manner other than through any remaining small peptide chains derived from purified proteoglycans. For this purpose,
The method of immobilizing the HSGAG substrate to the support may involve the oxidation of the sugar residues of the substrate (
For example, using periodate as described in the experimental protocol described below).

Assuming that the sample has heparanase activity, the method of step (i) involves the extent of heparanase activity in the sample, and the presence of HS in the presence of growth factors bound thereto.
It results in the cleavage of the HSGAG polymer substrate depending on its ability to cleave the GAG polymer substrate.

Any cleaved HSGAG polymer matrix (with both the first portion and the growth factor bound to it) is released into the analyte phase. Confirmation that HSGAG-growth factor is removed from the solid phase of step (i) is confirmed by washing with heparanase-treated solid phase after step (ii) and (strept) avidin-conjugating enzyme or anti-growth factor conjugating enzyme It is obtained by incubation with and by comparison of the signal with that obtained from the control solid phase. Heparanase activity clearly reduces the signal obtained.

In step (ii), the analyte phase can form a complex with the first binding moiety,
Alternatively, it is treated with a second solid phase support system that includes a second portion capable of forming a complex with a growth factor. As a result, complex formation between the first and second part or complex between the growth factor and the second part occurs, resulting in a cleavage fragment that becomes immobilized on the second solid support. . Either the first part or the growth factor remains unbound,
Used for signal generation.

In step (iii), a measurable signal is generated from the cleaved fragment immobilized on the second solid support. The signal may depend on the growth factor component of the complex (when the complex is captured by the first binding moiety and detected by anti-growth factor) and the enzyme bound to the anti-species antibody. Alternatively, if the complex is captured by an anti-growth factor antibody, the signal can be produced by an enzyme linked to a third moiety that can bind to the first moiety. The signal is, for example, a colorimetric signal and can be measured spectrophotometrically. Alternatively, the signal can be a fluorescent signal that facilitates automation of high throughput assays.

Finally, in step (iv), the signal generated in step (iii) is measured. This measurement is carried out in a second solid phase support system separated from the first such system (ie the one used in step (i)) and the signal is given in the environment of the first such system. Than measure).

The measured signal is indicative of the specific heparanase activity of the original sample. More specifically, the measured signal directly reflects the biological effect of producing the growth factor-HSGAG complex produced in physiological and pathological conditions.

A key control point of these pathologies is the bioavailability of key cytokines, chemokines and growth factors. When released from secretory cells, most (> 70%) of the mediators normally interact with HSGAG in the extracellular matrix (ECM), forming a stable reservoir of mediators. Only a few secreted mediators directly and indirectly bind to specific mediator receptors on target cells and initiate cell signaling. The heparanase enzyme releases these physiologically specific growth factors from the ECM and binds to target receptors,
Initiating signaling provides important control of this reservoir.

Cytokines that bind to HSGAG include IL-1α and IL-1β, IL-
2, IL-4, IL-7, IL-8, IL-12, TNFα, IFNα.
Growth factors that can bind to HSGAG include VEGF, FGF-1, FGF-2, and TF.
Includes Gβ1, PDGF, HGF. The term growth factor is used to mean any of these mediators. The present invention uses any of these heparan sulfate bound GFs to measure heparanase activity.

Heparanase-releasing growth factor in the form of a complex of HSGAG sequences of various lengths bound to growth factor is clearly more biologically active than natural growth factor.

Few binding sites in HSGAG that interact with these growth factors are currently described and each growth factor may have a unique binding site. Alternatively, certain growth factors may share certain binding sequences in HSGAG.

Heparanase from different cell sources has its ability to solubilize certain growth factors,
Or the combination of growth factors from HSGAG may differ from each other. This occurs where the growth factor protects where it binds to the HSGAG substrate or where the enzyme cleavage site sterically occludes. Thus, the use of a panel of growth factors that bind HSGAG may be able to confer on platelet-derived heparanase a specific pattern of GF solubility, as compared to spinal fluid or tumors. This can be important, for example, in differentiating the actions of tumor heparanase and platelet-derived heparanase.

Significant importance in the design of antagonists of heparanase function produces selective heparanase inhibitors of excellent specificity, such that inhibition is limited to the HSGAG cleavage site linked to certain growth factor solubilizations. Is the use of this method to:
Thus, for example, specific inhibitors of VEGF solubilization do not inhibit the immune function of platelet heparanase in the solubilization of IFNα to protect tumor and metastasis angiogenesis from infection during wound healing, Can be reduced and suppressed.

The assay is unaffected by the presence of proteases, hyaluronidases and chondroitinases. This assay is only sensitive if it requires a 30-60 minute incubation time of step (i).

The method of the invention may be used, for example, for the purpose of diagnosing or characterizing a particular medical condition,
It can be used to measure heparanase activity in biological samples. However, it is believed that an important application of the invention is the screening of potential inhibitors or enhancers of heparanase activity. Thus, for example, an assay can be performed with an analyte containing heparanase and its putative inhibitor to determine the effect of the inhibitor (or the opposite effect).

Preferred embodiments of the present invention are described below. The assay of the present invention is particularly useful for measuring heparanase activity, for example for the purpose of assessing potential inhibitors.

The GAG polymer substrate immobilized on the support is preferably heparan sulfate glycosaminoglycan.

The first solid phase support system (ie the system on which the HSGAG polymer substrate is immobilized) is preferably a microtiter well. Assay is immobilized HSG
This can be done using trays of microtiter wells with AG polymer substrates or larger arrays. The assay of the present invention may itself be carried out using automated equipment techniques for screening compounds for anti-heparanase activity. If microtiter wells were used as the first support system, the second solid support system could be a microtiter plate lid with a coated probe extending into each assay well, as well as a second support system such as NUNC It can be the TMS® TSP screening system or the FALCON® FAST system. In this case, step (i) and step (ii) of the assay are performed during the same incubation time. Alternatively, the second solid phase is another coated microtiter well,
Performing (i) and (ii) sequentially, requiring physical transfer of contents from the first well to the second well.

When the cleaved substrate is captured by the first moiety (on the second solid support), the first moiety can be biotin and the second moiety can be strept (avidin). In this case, the signal may be generated by means of anti-growth factor antibody and enzyme-linked anti-species antibody.

If the cleaved substrate is captured by the growth factor, the second part can be an anti-growth factor antibody. In this case, the first part is biotin and the signal can be produced by an enzyme linked to (strept) avidin (as in the third part above).

The method of the present invention may be performed using the following protocol. GAG polymer substrate is simply photobiotinylated with biotin.
n) Label using chemistry. Photobiotin (in phosphate buffered saline (PBS)
A mixture of 1 mg / ml) and HSGAG (10 mg / ml) at a ratio of 2: 1 (vol / vol) was subjected to a microscope UV.
10 cm to UV light emitted from Mercury arc HBO50 W lamp used as source
Exposure for 21 minutes at a distance of. Biotinylated HSGAG is dialyzed against PBS for 18 hours at 4 ° C and stored at -20 ° C.

Immobilization of the GAG polymer substrate onto the solid support is performed after labeling with the first complex-forming moiety, the substrate is oxidized with periodate, and the carbohydrate-binding microtiter well (eg, COASTER ( (Registered trademark) or NUNC plate can be used) to create a surface to which the substrate is covalently and stably bound. Specifically,
10 μM sodium periodate and 5 μg / ml biotinylated HSGAG in sodium acetate buffer (10 mM, pH 4.0) were allowed to interact for up to 15 minutes, then 1
Add 00 μl of activated biotinylated HSGAG to each well of the carbohydrate binding assay plate for 1 hour and shake at 37 ° C. The plates are then washed extensively with PBS containing 0.1% Tween® 20. 100 μl of 1% bovine serum albumin (BSA) in PBS is added to each well and incubated at 37 ° C. for 60 minutes to block non-specific binding on the plate. Following washing with PBS, the coated plates can be used immediately or stored in polyethylene wrap at 4 ° C for several months.

Once the GAG polymer substrate is immobilized on the first support, it (ie the substrate) is GA
It can be treated with a G-coupled growth factor (eg VEGF, TGFβ1, MGP-1, IFN-γ, see list in claim 8). 1-5 μg / ml GF in 100 μl PBS
Is added to each well and incubated for 18 hours at 4 ° C. Following washing with PBS, the plates are ready for use.

The second solid phase support may be prepared by coating with a support material with a second complex-forming moiety such as BSA-blocked (strept) avidin.
If the second solid phase is another well, add a microwell lid probe or well,
Coat with 1-5 μg / ml (strept) avidin in PBS for 18 hours, followed by P
Wash with BS and they will be blocked with BSA as above.

To perform the assay, the analyte having (or possibly having) heparanase activity is incubated in the presence of the first solid phase support system. The analyte is diluted in enzyme assay buffer which is Ca ²⁺ (5 mM), Cu ²⁺ (1 mM) and Fe ³⁺ (1 mM) containing sodium acetate (0.1 M, pH 5.5). Each assay contains, as controls, wells without biotinylated HSGAG coating and wells that received no test sample. Heparanase assays are usually
1 hour (but can range from 30 minutes to 4 hours), 37 on plate shaker
Proceed at ℃. If the second solid phase is a well-covered probe, then steps (i) and
If (ii) is performed simultaneously and the second solid phase is another coated well, the supernatant from the first well is transferred to the second well and incubated for 2 to 24 hours.

As a result, the biotinylated HSGAG-GF substrate cleaved during the incubation from the first solid support is immobilized on the second support, where it can be separated from the analyte.

Next, a signal is generated. If the second solid phase used strept (avidin) to capture the complex via its biotin moiety, this can be achieved by incubation of the second support with anti-growth factor followed by enzyme-conjugated species IgG. . When the second solid phase uses an anti-growth antibody to capture the complex, the signal can be generated by incubation with enzyme-conjugated strept (avidin). The cleaved configuration of the substrate bound to the first solid phase can be obtained by incubation with enzyme-conjugated strept (avidin) or enzyme-conjugated antibody. The preferred enzyme conjugate for this detection reagent is peroxidase, therefore it will be apparent to use the amount of peroxidase activity associated with the wells in standard TMB substrate color change and spectrophotometric methods.

The invention is illustrated by the following non-limiting examples and reference examples, and the accompanying figures illustrating the results of reference examples 1 and 2.

Reference Example 1 This reference example demonstrates the properties of biotinylated HSGAG for heparanase. Biotinylated HSGAG was prepared using the method described in the protocol above and immobilized in microtiter wells as a solid phase support also using the method of the protocol.

Separate samples of support-bound biotinylated HSGAG were incubated at 37 ° C. for 1 hour at very high concentrations (> 100-fold than encountered in clinical samples) of the first concentration of protease shown in Table 1 below. , Incubated with various enzymes including, for example, dispase, thrombin, collagenase, trypsin, plasmin, pepsin and glycosidases such as neuraminidase and endoglucoronidases such as heparanase, hyaluronidase and chondroitinase.

[0053]

[Table 1]

After the first incubation time, the% release of HSGAG was measured by the following method. 100 μl of avidin peroxidase diluted 1: 1000 in PBS was added to the plate for 60 minutes at 37 ° C. After washing 3 times with PBS Tween, 100
After addition of μl TMB substrate (100 μg / ml) and quenching the reaction with 100 μl 0.1M HCl, color development was assayed at 450 nm in endpoint mode.

The above method was repeated with all the above enzymes using a dilution factor of 400 to 800 compared to the initial concentration. The results are plotted in the attached FIG. The plot in Figure 1 clearly demonstrates the properties of biotinylated HSGAG for heparanase.

Only hyaluronidase, dispase and thrombin have a slight effect on the substrate, whereas in the case of dispase and thrombin they disappear on dilution (FIG. 1). Collagenase, trypsin, plasmin, pepsin, chondroitinase and neuraminidase had no discernible effect on the substrate, with% release too low for these enzymes to be plotted graphically.

Reference Example 2 This reference example demonstrates that support-bound biotinylated HSGAG is cleaved from the solid phase by heparanase treatment. Biotinylated HSGAG immobilized on the solid phase was prepared by the above protocol.

A dilution series of heparanase spleen extract was prepared and added to separate samples of support-bound HSGAG substrate and incubated for 60 minutes. The support is then washed, then avidin peroxidase 1: 10000 for 30-60 minutes at 37 ° C.
Added in. The peroxidase substrate TMB 100 μg / ml was added and the color change was measured spectrophotometrically.

The results are plotted in Figure 2, which demonstrates that the loss of biotinylated HSGAG is proportional to heparanase dilution over two orders of magnitude.

Reference Example 3 This reference example demonstrates that the complex of HSGAG and growth factor is cleaved from the solid phase. Biotinylated HSGAG prepared as in the protocol above was immobilized on a solid phase using the method in the protocol above.

Recombinant growth factor / cytokine (63 ng / ml) was incubated in the wells overnight to allow HSGAG binding and solid phase binding complex formation. Unbound GF / cytokine was removed by washing 3 times with PBS.

A standard dilution of spleen heparanase enzyme 1:20 was added to the wells for 1 hour, leaving the remaining G
F / cytokine potency was measured using specific anti-GF / cytokine antibodies. The antibody was detected by anti-species peroxidase binding reagent followed by substrate development.

[0063]   The results are shown in Table 2 below.

[Table 2] This result demonstrates heparanase cleavage from the solid phase of the complex of HSDGAG and GF / cytokine.

Example 1 This example demonstrates the detection of biotinylated HSGAG-growth factor complex. Biotinylated HSGAG prepared as in the protocol above was also immobilized on a solid phase using the method of the protocol.

Recombinant VEGF (63 ng / ml in PBS) was incubated in the wells overnight and
SGAG was allowed to bind to form a solid phase binding complex of HSGAG and VEGF.

Unbound VEGF was removed by washing 3 times with PBS. Heparanase enzyme from spleen diluted 1:10 was added to the wells for 1 hour. Subsequently, the contents of the wells were pre-coated with a monoclonal anti-VEGF antibody (5 μg
/ Ml for 16 hours) and transferred to another ELISA plate and incubated for 4 hours.
During the incubation, biotinylated HSGAG heparanase cleavage soluble complex bound to VEGF, which complex is from the first plate, was bound to anti-VEGF coated plates. After washing, the presence of biotinylated HSGAG bound to anti-VEGF wells was measured by the addition of avidin peroxidase followed by a peroxidase substrate.

The results are shown in Table 3 below, which also includes the background and control experiments detailed in the table.

[Table 3]

From the above results, the method of the present invention shows that the anti-V of VEGF and anti-biotinylated HSGAG
It is seen that the capture of the cleavage complex by the EGF antibody is accompanied by the detection of the cleavage complex by avidin peroxidase.

[Brief description of drawings]

FIG. 1 is a graph demonstrating the properties of biotinylated HSGAG for heparanase.

FIG. 2 is a graph demonstrating that support-bound biotinylated HSGAG is cleaved from the solid phase by heparanase treatment.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] July 3, 2001 (2001.7.3)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, C H, CN, CR, CU, CZ, DE, DK, DM, DZ , EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, K G, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, S D, SE, SG, SI, SK, SL, TJ, TM, TR , TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Paul Ernest Charles Bren             Chilly             Cheshire, M33.5 Edy, England             ー, Sale, Dunchurch No. 5 F-term (reference) 2G045 AA40 DA20 FB01                 4B029 AA07 FA12                 4B063 QA01 QQ38 QQ79 QR18 QR48                       QR82 QX02

Claims (21)

[Claims] 1. (i) a sample containing immobilized HSGs for heparanase
Incubating in the presence of a first solid support having an AG polymer substrate thereon, wherein the substrate is insensitive to the action of a protease, the substrate having a first binding moiety attached thereto, and A paracrine cell regulator capable of binding to the HSGAG bound thereto, (ii) a second portion having a second portion provided on the incubated sample
Incubation with a solid support where the HSGAG polymer substrate can be cleaved from the first solid support to the second support by attaching the second moiety to a paracrine cell regulator or a first binding moiety. (Iii) a measurable signal is generated on the other of the first or second portions of the cleaved substrate immobilized on the second supporting solid phase, and (iv) the second solid phase separated from the first solid phase support. Assaying a sample for measuring heparanase activity comprising measuring the signal on a phase support. 2. The method of claim 1, wherein the HSGAG polymer substrate is covalently attached to the first solid support system. 3. The method of claim 1 or 2, wherein the first solid support system is a microtiter well. 4. The method of claim 3, wherein the second solid support system is a plate lid probe. 5. The method of claim 3, wherein the second solid support system is a microtiter well. 6. The method according to any of claims 1 to 5, wherein the second solid support system is separated from the first solid support system between steps (i) and (ii). 7. The HSGA cleaved from the first solid support in step (ii).
7. The method of any of claims 1-6, wherein the G polymer substrate is immobilized on the second solid support by the attachment of the first and second moieties. 8. The method of claim 7, wherein the first moiety is biotin and the second moiety is strept (avidin). 9. The method according to claim 7 or 8, wherein the signal is generated by an enzyme bound to the anti-paracrine cell regulator antibody and the anti-species antibody. 10. The method of claim 9, wherein the signal is generated by incubation of the second solid support with an anti-paracrine cell regulator antibody followed by a peroxidase anti-species IgG. 11. The HSGA cleaved from the first solid support in step (ii).
The G-polymer substrate is linked to the paracrine cell regulator and the second part by conjugation to the second part.
The method according to claim 1, wherein the method is immobilized on a solid support. 12. The method of claim 11, wherein the signal is generated by the binding of an enzyme with a third moiety that can bind to the first moiety. 13. The method of any of claims 1-12, wherein the paracrine cell regulator is a growth factor, cytokine or chemokine. 14. The paracrine cell regulator is a growth factor and VEG
F, TGFβ1, FGF-1, FGF-2, HGF, PDGF, IL-1α, I
L-1β, IL-2, IL-4, IL-7, IL-8, IL-12, IP-10
The method according to claim 13, wherein the method is selected from the group consisting of: 15. The method according to any one of claims 1 to 14, for measuring the activity of a putative inhibitor of heparanase activity. 16. An HSGAG polymer substrate immobilized thereon having a first portion bound thereto and a paracrine cell regulator capable of binding HSGAG, the first portion or the paracrine cell regulator being capable of binding a second portion. A solid support having 17. The support of claim 16, wherein the paracrine cell regulator is a growth factor, cytokine or chemokine. 18. The paracrine cell regulator is VEGF, TGFβ1,
FGF-1, FGF-2, HGF, PDGF, IL-1α, IL-1β, IL-
2, IL-4, IL-7, IL-8, IL-12, IP-10, MCP-1, T
18. The support of claim 17, selected from NFα or IFN-α. 19. The support according to claim 16, wherein the first portion is biotin. 20. A combination of a solid phase support according to any one of claims 16 to 19 and a second solid phase support on which the second portion is provided. 21. An assay kit comprising the combination according to claim 20 and a signal generating agent for generating a signal indicating the result of the assay.