JPS62265998A - Heparan sulfate endoglycosidase assay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an assay method for endoglycosidase enzymatic activity and a labeled substrate used in the assay method.

The assay method of the present invention is expected to be useful in detecting the malignancy of cancer.

Certain biological substances called proteoglycans are
Extracellular matrix of connective tissue forms the basic substance in IJ socks. These proteoglycans are high molecular weight polyanionic substances that contain various types of heteropolysaccharide side chains covalently bonded to the polypeptide backbone. These proteoglycans contain more than 95% carbohydrate. This polysancharide group of proteoglycans was previously called mucopolysaccharides, but now they are often called glycosaminoglycans because they all contain glycosamine or galactosamine.

Many enzymes are involved in the metabolic degradation of normal proteoglycans. The initial proteoglycan degradation is often proteolysis, which separates or digests the protein components. This protein breakdown produces glycosaminoglycans. The glycosaminoglycans, in turn, generate smaller glycosaminoglycan fragments through the enzymatic activity of glycosaminoglycan endoglycosidase.

Furthermore, glycosaminoglycans or fragments thereof generate monosaccharides from the non-reducing ends of the glycosaminoglycans through the enzymatic activity of glycosaminoglycan endoglycosidase.

Interest in endoglycosidases has recently increased due to the potential association of these enzymes with tumor invasive and metastatic activity. Nicholson (N1colson)
) (Biochem, Biophys, Acta (Bioc)
hem, Biophys, Acta), 19
(1982, Vol. 695, pp. 11"3-176) reviewed various oligosaccharide-degrading enzymes (141
(pages 142 to 142) have been reported to be associated with malignant diseases. Nicholson (N1colson) (Journal・
Of Histochem and Cytochem.

U, Histochem, & Cytochem,
) (1982, Vol. 30, pp. 214-220) describe the mechanism of tumor cell invasion of the endothelial cell basal layer and the production of associated degradation products derived from proteins and glycosaminoglycans.

Kramer et al. (Journal of Biological Chemistry (J, Biol, Ch.
em, ) 1982, vol. 257, 2678-2686
(Page 1) report on a tumor-derived glycosidase that can specifically cleave glycosaminoglycans and release fragments enriched in heparan sulfate.

Irimura, etc. (Analyt, Biochem,
), Vol. 30, pp. 461-468) describes high performance gel permeation chromatography of glycosaminoglycans. The heparan sulfate degrading activity of melanoma cells is measured using this chromatographic technique.

Nakajima etc. (Science (5c)
220, pp. 611-613.

(1983) described the relationship between metastatic activity and heparan sulfate-degrading activity in melanoma cell lines.

The disappearance of high molecular weight heparan sulfate was followed by polyacrylamide gel electrophoresis, staining and deencytometry.

Vlodavsky et al. (1983)
Cancer Research (Caner Res, )
43, pp. 2704-2711) describe the degradation of 35S-labeled proteoglycans from convergent endothelial cells by two T-lymphocyte cell lineages. The highly metastatic cell lines have higher 35S release activity than the less metastatic lines.

Irimura et al. (1983, Proceedings in American Society of Cancer Research) (Proc, Am.

Soc, Cancer, Res,) Volume 24, 37
Page, Abstract 144) describes the heparan sulfate-degrading enzyme activity of melanoma cells using high-performance liquid chromatography. Nakajima etc. (
1984, Journal of Biological Chemistry (J, Biol, Chew,) 259
Vol. 2283-2290) describe the properties of metastatic melanoma heparanase. High performance gel permeation chromatography and chemical analysis were used to describe the functional substrates and the products generated.

The background described herein includes an interest in simple, accurate, and reproducible endoglycosidase assays, especially since endoglycosidases play a critical role in the establishment of tumor metastase.

The ability of tumor cells to invade host tissues and metastasize to distant, often specific organ sites is one of their most important properties. Metastasis formation is a series of unique interactions between tumor cells and normal host tissues and cells. These processes include invasion of surrounding tissue, lymphatic penetration of blood vessels and transfer with lymph or blood, or dispersion into sparse cavities, capture and invasion at distant sites, and proliferation to survival and secondary lesion formation. consists of a number of individual and selective steps.

The basement membrane is a continuous membrane of extracellular matrix containing collagen and non-collagen proteins and proteoglycans that separates the cells of the parenchyma from the underlying interstitial connective tissue. They have a characteristic permeability and play a role in maintaining tissue structure. Tumor cells that metastasize must invade epithelial and endothelial basement membranes during invasion and metastasis. Invasion and destruction of the basement membrane by the invading tumor cells can be observed using an electron microscope. Because the basement membrane is a rigid structure formed by a unique combination of macromolecules including type collagen, laminin, heparin sulfate (H3), proteoglycans, and fibronectin, successful entry into the basement membrane probably requires one or more requires the active participation of enzymes derived from tumor cells.

Due to its physical and chemical properties such as polyanionicity and protection against macromolecules (Kanwar),
war) et al., 1980, Journal of Cellular,
Biology (J, Ce1l.

Heparin sulfate (H3) is an important structural element of the basement membrane.

H3 binds to fibronectin, laminin, and collagen, and these molecules are observed collectively in the basal layer by using antibodies raised against individual elements. H3 can be included in the basal lamina matrix assembly by promoting interaction with collagen and non-collagen proteins while protecting them against proteolytic attack. Thus, disruption of the HS proteoglycan barrier is important for invasion of the basement membrane by tumor cells.

The interaction between malignant cells and vascular endothelium has been studied using a monolayer of cultured vascular endothelial cells with a synthetic extracellular matrix that mimics basement membrane. Using this model, metastatic B.
16 melanoma cells were found to degrade matrix glycoproteins such as fibronectin and matrix sulfated glycosaminoglycans such as heparin sulfate.

It has been proposed that metastatic tumor cells characteristically possess heparin sulfate endoglycosidase properties, since heparin sulfate is released into solution as fragments approximately one-third of its original size.

The relationship between the metastatic potential and the ability to enzymatically degrade the extracellular subendothelial matrix of the five 816 melanoma sublineages with different transplantation and invasive properties shows that the more invasive and metastatic 816 sublineages have the lower metastatic potential. Nakajima et al., 1983, Science (5science);
220, p. 611), and B16 cells themselves (or cell-free homogenates), which have a high ability to form lung colonies, also degrade purified heparan sulfate at a higher rate than B16 cells, which have a lower ability to form lung colonies. (Id.) studied the ability to degrade H3 and other purified glycosaminoglycans of various origins (heparin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid). For analysis of glycosaminoglycan degradation products, high performance gel permeation chromatography (Irimura) was used.
et al. 1983, Analytical Biochemistry
(Aral, Biochem,) Vol. 130, p. 161,
Nakajima et al., 1984, Journal of Biological Chemistry (J.
Biol, Chem, Vol. 259, p. 2283).

(35s) H3 metabolically labeled with sulfuric acid was extracted from the subendothelial matrix of EH3 sarcoma and PYS-2 carcinoma cells, which form the basement membrane, and middle aortic artery endothelial (BAE) and corneal endothelial (BCE) cells. Purified (ibid.).

83 purified from live lung and other glycosaminoglycans
The molecule was labeled with tritium at its reducing end using NaB('H)4. These labeled glycosaminoglycans were incubated with B16 cell extracts in the presence and absence of D-sugar acid 1,4-lactone, a potent exo-betacurcuronidase inhibitor, and their degraded fragments was analyzed by high performance gel permeation chromatography.

H3 isolated from the various sources mentioned above is all broken down into fragments of characteristic molecular weight, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin, which are essentially not degraded. Disassembled. Unlike other glycosaminoglycans, heparin inhibited the degradation of H3. The time dependence of H3 degradation into specific molecular weight fragments in melanoma, heparanase
was shown to cleave at a specific intrastrand site. (Same magazine)
To determine its specific H3 cleavage site, the newly generated reducing end of the H3 fragment was cut into (1) NaB ('H)
(2) hydrolysis to monosancalides and analysis of these saccharides by paper chromatography. Since the 3H-reducing terminal monosancharide derived from the H8 fragment is overwhelmingly (>90%) L-gulonic acid, the H3-degrading enzyme responsible for this reaction was endoglucuronidase (heparanase). .

H3-degrading endoglucuronidase is found in rice tissues such as human skin fibroblasts, rat liver cells, human placenta and human platelets. H3-degrading endoglucuronidase in mammalian cells was previously described by other researchers as a “heparitinase” directing heparitin sulfate (heparan sulfate)-specific endocricosidase.
It was reported that. However, heparitinase was originally produced by Flavobacterium heparinum (Flavobacterium heparinum).
Eliminating enzyme (e-rium heparinum)
EC4,2,2,8), and this enzyme is a non-sulfate and monosulfate 2-acetamide-
Cleavage of 2-deoxy-alpha-D-glucosyl-D-hexuronic acid. H3-specific endoglycosidases in mammalian cells, other than those found in skin fibroblasts, are endoglucuronidases;
The endoglucuronidase of mammalian cells that is capable of decomposing

Glycosaminoglycan endoglycosidases have been assayed for their enzymatic activity by several other methods. For example, Oldberg
) etc. (1980, Biochemistry (Bio c
Hem., Vol. 19, pp. 5755-5762) describes an assay for platelet endoglycosidase, which degrades heparin-like polysaccharides.

This assay involves measuring the amount of reduction in 3H-heparan sulfate. This amount of decrease correlates with endoglycosidase activity.

An endoglycosidase assay using a solid-phase substrate is described by Iverius (1971, Biochem, J.) 1
24, pp. 677-683) and Oosta (Oosta
) etc. (19, 1982, Journal of Biological Chemistry (J, Biol, Chem, )
257, pp. 11249-11255).

Iverius coupled various glycosaminoglycans to Sepharose 4B beads activated with cyanogen bromide. In some cases, the endoglycosidase hiruronidase was assayed for enzyme activity by incubating the enzyme with chondroitin sulfate bound to Sepharose 4B. The enzyme activity was monitored by tracking soluble uronic acids in a colorimetric assay. Oosta etc.
An assay for one platelet-derived endoglycosidase, heparitinase, which cleaves heparin and heparan sulfate is described.

The Oosta et al. system and assay includes:

(11 heparin and N-succinimide 3- (4-1=
Combination with droxyphenyl) propionate.

(2) Combined heparin with Na”SI and chloramine-T
Labeling by incubating with.

(3) Binding of I2J-labeled heparin to cyanogen bromide-activated Sepharose 4B pieces.

(4) incubating endoglycosidase with Sepharose 4B pieces bound with 1'I-labeled heparin;
Measure the solubilized radioactivity.

In these two methods, glycosaminoglycans are
The free amino groups of the glycosaminoglycans are cross-linked to agarose by reaction with amino-reactive cyanogen bromide-activated agarose. Since glycosaminoglycans such as heparin and heparan sulfate have several free glucosamine amino groups, this type of cross-linking results in excessive covalent bonding between the substrate molecule and the agarose gel. This results in loss of endoglycosidase capacity and non-linear degradation rates. Thus, the most desirable solid phase substrates for glycosaminoglycan endoglycosidases are glycosaminoglycans with one end of the molecule, such as the reducing end, attached to a solid phase support.

Included in the invention are solid phase substrates that produce soluble labeled products upon hydrolysis by glycosaminoglycan endoglycosidases, and methods for producing such substrates. This solid phase substrate contains a glycosaminoglycan with a labeled N-acetyl group and its reducing end reductively aminated to create an amine terminus. Furthermore, the substrate is coupled via its amino terminus to the amine-reactive solid matrix.

The method of making a solid phase substrate includes the following steps. (b) At least some of the glycosaminoglycans are N-desulfated or N-acetylated. (B) At least some of them are labeled with N-desulfated or desulfated glycosaminoglycans. (c) Completely N-acetylate the labeled glycosaminoglycan using acyl anhydride or acyl halide. (d) The above labeled glycosaminoglycan. reductively aminating the reducing end of the molecule to produce a labeled amino-terminated glycosaminoglycan. (e) Transfer the labeled amino-terminated glycosaminoglycan to an amino-reactive solid phase support via its amino terminus. Combine to create a solid matrix substrate.

Labeling is accomplished by displacement of the amine groups of the partially N-desulfated or N-acetylated glycosaminoglycans by some substance to produce a detectable signal. This substance includes a radioisotope label, a fluorescent label, or an enzymatic label. Fluorescent labels are preferred for ease of assay and radioactive labels for their similarity to natural glycosaminoglycans.

The present invention relates to a new assay for glycosaminoglycan endoglycosidase activity, and in particular to that of a heparan sulfate endoglycosidase called "heparan sulfate." This new assay describes the use of a solid phase substrate that yields a soluble labeled product upon hydrolysis by glycosaminoglycan endoglycosidases. The assay also states that this solid phase enzyme assay can also be applied to liquid phase conditions.

For example, 1 such as heparan sulfate endoglycosidase
Immunoassays for measuring glycosaminoglycan endoglycosidases, such as those using antibodies raised against the enzymes, have also been described.

This solid phase substrate contains glycosaminoglycans with radiolabeled N-acyl groups. These labeled N-
Although other labeled acyl groups such as formyl group and propionyl group can be used as the acyl group, 3H-labeled or +40-labeled acetyl group is preferable. The solid phase substrate of the present invention includes hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate,
Contains dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or a combination thereof. The use of special glycosaminoglycans allows assays for the enzymatic activity of endoglycosidases with substrate specificity for the particular glycosaminoglycan used.

The amino-reactive solid matrices to which amino-terminally labeled glycosaminoglycans are attached have many acceptor types, both in the fundamental nature of the matrix and in the amino-reactive chemical sites. Preferred solid matrices are agarose-based, most preferably Sepharose or Sepharose derivatives in the form of beads (Pharmacia). Other solid matrices such as cellulose and polyacrylamide can also be used if they have amine-reactive substituents for attachment.

Sepharose beads are activated with cyanogen bromide and are well known to bind amine-containing molecules such as heparin, other glycosaminoglycans or glycosaminoglycan derivatives. Coupling via cyanogen bromide is a very useful coupling method in the practice of this invention. Cyanogen bromide-activated agarose or other amine-reactive solid matrices bind one or more amine groups of glycosaminoglycans and glycosaminoglycan derivatives, which have many amino groups. This multiple bond to the labeled glycosaminoglycan is
Since hydrolysis reactions by a single glycosidase do not result in a single soluble product, e.g., the production of a product that is not bound to a solid matrix,
It can be said that this is a very insensitive and inaccurate glycosaminoglycan endoglycosidase assay. Thus, in the practice of the present invention, it is important that the labeled glycosaminoglycan derivative attached to the solid matrix has only one primary amino group.

Although many amine-reactive substituents are known to those skilled in the art, N-hydroxy succinide ester is the preferred amine-reactive group attached to the solid matrix, and is commercially available and easily synthesized. The N-hydroxy succinide ester binds to primary amine groups at a pH between about 6 and 9. Agarose can be activated to have aldehyde groups by periodate oxidation. This aldehydic agarose can react with labeled amine-terminated glycosaminoglycans, and the bond is stabilized by reduction with sodium cyanoborohydride.

(Perikh, etc.Methods in Enzymology,)X
XX Volume IV, page 81 Academy Press (Acad.

(1974)) Other common techniques for attaching amine-containing labeled glycosaminoglycans to solid matrices include (1) carbodiimides and carboxyl-containing solid matrices, (2) bromoacetyl,
Examples include directly reacting a solid matrix containing diazonium or epoxy groups with an amine-containing labeled glycosaminoglycine.

Generally, glycosaminoglycans have sulfated or acetylated amine groups. For example 1, after at least partial N-desulfation or N-acetylation,
Primary amino groups generated on glycosaminoglycans can be used for labeling. Deacetylation can be carried out by hydrazinolysis under conditions that avoid excessive alkalinity leading to hydrolysis of glucosaminyl bonds. Desulfation is carried out by formation of pyridinium salts of glycosaminoglycans followed by solvolysis with dimethyl sulfate.

Amino group labeling can be accomplished by reaction with a fluorescent compound such as fluorescein isothiocyanate, an enzyme (and bifunctional coupling reagent) such as alkaline phosphatase, or a radiolabeled acyl anhydride or halide. The label is covalently bound to at least some of the free amine groups. The remaining free amine groups of the labeled glycosaminoglycan are acetylated, for example, by treatment with acetic anhydride.

The acylated labeled glycosaminoglycan is further aminated at its reducing end. This amination is carried out by incubation with an amine salt to form a terminal Schiff base and further reduction to form a terminal amine.

Labeling of amine groups is carried out by attaching measurable compounds or active enzymes to at least some of the amino groups. The measurable compound may be one of many known to absorb visible light very well, or preferably fluoresce upon irradiation with light at a particular wavelength, such as fluorescein mentioned above. It is one of the things. Labeling by binding of enzymes as active proteins (such as the alkaline phosphatase mentioned above) to partially N-desulfated or N-acetylated glycosaminoglycans is also one possibility. However, labeling with enzymes or measurable compounds with light-absorbing or fluorescent structures will involve sterically bulky substitutions. When it is oversubstituted, the sterically bulky substitution creates a glycosaminoglycan derivative that is unfavorable as a substrate for glycosaminoglycan endoglycosidases. In a preliminary experiment,
Partially N-desulfated heparan sulfate is combined with fluorescein inthiocyanate in a 1:1 ratio. This fluorescein-labeled derivative was found to be a good substrate for melanoma heparanase. 10 for H3
It is believed that a conjugate of fluorescein is produced at a ratio of 1 to 1 and can serve as a substrate for heparan sulfate.

With one or several such bulky substitutions,
Substrate activity is not interfered with and a well labeled substrate is provided. Another significant problem with enzyme labels is that enzymes generally possess free amine groups that become bound to amine-reactive solid matrices.

One preferred label for glycosaminoglycans is
A radiolabel that is structurally similar or identical to a naturally occurring N-substitute. While 3SS-sulfated N-substitutes are utilized, +40 or 3H-acetylated N-substitutes are also preferred for ease of production. Although the substrates and procedures described below primarily concern radiolabeling, other labels, especially fluorescent labels, are very useful in principle.

When an N-radiolabeled glycosaminoglycan is attached at one end to a solid matrix by a single bond, a solid phase substrate for glycosaminoglycan endoglycosidase is generated. As described elsewhere, this solid phase substrate yields a soluble radiolabeled material through the action of the enzymatic activity of glycosaminoglycan endoglycosidase. Another way to measure this same activity is to also observe the disappearance of radiolabel bound to the solid matrix due to enzymatic activity. This type of measurement is a negative measurement method;
and has the disadvantage that the incubation supernatant must be carefully removed from the remaining solid matrix substrate.

In a broad sense, the solid phase substrate of the present invention is one that upon hydrolysis by glycosaminoglycan endoglycosidases yields a soluble material labeled with a detectable signal. The solid phase substrate contains a glycosaminoglycan with a label that does not interfere with hydrolysis of the labeled glycosaminoglycan by glycosaminoglycan endoglycosidase. The labeled glycosaminoglycan is attached to the solid matrix through a single end, preferably through the reducing end, and with a single covalent bond. Detectable signals include radioactivity, optical absorption, fluorescence, enzymatic activity, and the like. The solid matrix will preferably be hydrophobic and will include polymers such as cellulose, dextran, polyacrylates and derivatives thereof, alone or in combination. The substrates of the invention are soluble if a detectable label is present with the bound molecule. The binding molecule can be used as a "handle" to remove a portion of the bound glycosaminoglycan.

For labeling at least partially desulfated or deacylated glycosaminoglycans, treatment with 3H-acetic anhydride or 14C-acetic anhydride, although analogous acetyl halides, especially chlorides or alkyl bromides, may be effective. This can be done best by. In addition to other acyl groups such as formyl or propionyl, other attachment methods can also be used for this labeling procedure.

The substrates of the present invention can also be liquid phase substrates due to the separation of the cleavage products from the uncleaved substrate after the enzymatic reaction. Within this scheme, a glycosaminoglycan, such as heparan sulfate, could be attached at one end, preferably the reducing end, to another molecule. The glycosaminoglycans are labeled at other sites with other molecules such as 12J, fluorescein, enzymes and the like, which are used to detect the cleaved products during the assay.

The advantage of using a liquid substrate of the type described here is that it is a sensitive assay for the action of glycosaminoglycan endoglycosidases. This increased sensitivity is at least related to the increased availability in solution for soluble enzymes.

It appears that the molecular label at one end of the glycosaminoglycan can be a small molecule, such as fluorescein or biotin, or a larger molecule, such as a peptide or protein. Attachment of this molecule to one end of the glycosaminoglycan does not significantly inhibit hydrolysis of the labeled glycosaminoglycan by glycosaminoglycan endoglycosidases. This molecular beacon must have the ability to act as an important "handle" for the labeled glycosaminoglycan chain and for the glycosaminoglycan chain residues that remain after cleavage by the glycosaminoglycan endoglycosidase.

As a "handle," the molecule can serve as a point of attachment for a protein molecule that has an affinity for the bound label molecule. Such protein-molecule interactions easily separate the molecularly labeled portion of the labeled glycosaminoglycan from the labeled but unlabeled portion generated by endoglycosidase hydrolysis of the glycosaminoglycan substrate. I think you can.

A molecular label is a) a hapten molecule capable of producing specifically binding antibodies when conjugated to a carrier such as a protein and immunogenically administered to an animal; b) a protein or peptide. C) part or whole of an immunogenic substance such as C) e.g. avidin or protein A;
It should be one of the substances that have a high affinity for the existing protein molecules, such as

After incubation with a sample containing endoglycosidase activity, the uncleaved products can be separated from the cleaved products by incubation with, for example, a solid-phase antibody that has an affinity for the molecular label.

Proteins other than antibodies that bind to molecular markers attached to the ends of glycosaminoglycans can also be used to separate uncleaved glycosaminoglycans. If a solid-phase antibody or solid-phase binding protein is used, the solid phase may be any support that readily binds or adsorbs the antibody or binding protein and can separate the cleaved substrate from the uncleaved substrate. Examples of commonly used solid phases include agarose, cellulose, sepharose, polymers such as polystyrene, glass and glass beads, magnetic beads, and the like. The solid phase can take the form of large or small particles, tubes, microplates, or other elements that are easily adapted to detection systems.

Separation of cleavage products from uncleaved glycosaminoglycan products can also be achieved by immunoprecipitation reactions, which do not require binding of antibodies to a solid phase (Morg
an) et al., 1962, Proceedings in Society of Experimental Biology.
Antomedicine (Proc, Soc, of E
xp, Biol, Med,) Volume 110, 29-3
(See page 5). Precipitating antibodies are directed against molecules labeled at the ends of glycosaminoglycan chains.

To enable terminal attachment to a solid matrix,
Further modification of labeled glycosaminoglycans is required. This modification is the substitution of a primary amino group at one end of the labeled glycosaminoglycan.

A preferred method of making this substitution is to incubate the labeled glycosaminoglycan with an ammonium salt and sodium cyanoborohydride under alkaline pH. First, a Schiff base is formed between ammonia and the aldehydic carbonyl group of the terminal hexose. This Schiff base is reduced to a -grade amino group by sodium cyanoborohydride.

Generally, the synthetic steps to produce the solid phase substrates of the invention include (
1) partial N-acylation, e.g. by hydrazinolysis, or N-desulfation, e.g. by solvolysis in dimethyl sulfoxide; (2) N-acyl by labeled acyl anhydrides or halides, for radiolabelling. (3) a reductive amination step; (4) attachment to the solid matrix through the newly introduced terminal amine;
Contains. To ensure that no amine-reactive functional groups remain on the solid matrix, the final step includes adding sodium cyanoborohydride and a compound with free amino groups to the matrix-labeled amine-terminated glycosaminoglycan. It is desirable to incubate the conjugated product. This latter compound is, for example, ethanolamine or glycine ethyl ester.

The substrates and procedures of the present invention exhibit a number of advantages for assaying glycosaminoglycan endoglycosidase enzyme activity.

For example, the substrates of the invention are attached to a solid matrix via a single carbohydrate-linked amino ligand and produce a linear pattern of enzyme products.

In the past, proteoglycans, including glycosaminoglycans, as well as bound protein components, were bound to solid matrices of agarose activated with cyanogen bromide. The proteoglycan appears to be attached to the agarose essentially through its protein component. Thus, proteolytic activity as well as glycosaminoglycan endoglycosidase activity generates soluble products. The specificity of the endoglycosidase enzymatic activity assay is less than the results shown by the present invention.

Heparan sulfate obtained from human melanoma cell lines (
It was found that heparan sulfate endoglycosidase) only partially degrades N-sulfate, deacetylated heparin. A similar enzyme preparation is N-desulfated N-
Similar to acetylated heparan sulfate, N-sulfuric acid was found to be efficiently cleaved into characteristic degradation fragments.

While there are many glycosaminoglycan endoglycosidases, heparan sulfate endoglycosidases or heparan sulfate, endoglycosidases that use heparan sulfate as one preferred substrate were selected as exemplary to illustrate the preferred embodiments of the invention. In addition, N-hydroxysuccinide agarose derivatives were selected as a preferred solid matrix to attach labeled amine-terminated heparan sulfate for the purpose of creating a solid phase substrate. Heparanase activity produced a radiolabeled product as specifically described in a number of the examples below.

Melanoma heparanase is one endo vector glucuronidase that cleaves H3 at a specific intracellular site. Figure 8 shows such melanoma heparanase specificity.
It was shown to. Thus, separation of the reaction products from the substrate (based on their size) is necessary in the heparanase assay; previously established methods such as polyacrylamide gel electrophoresis and high performance liquid chromatography Although effective for fragment characterization, it is not suitable for rapid quantitative assays of large amounts of samples; covalently bound substrates are required for rapid quantitative assays.

The solid-phase assay substrate developed here is Affigel (Af
fi-Gel) 15, partially N-acetylated or N-desulfated, N-MH or 14C]-acetylated H3.

In this substrate, the H3 derivative is attached to agarose through only one covalent bond (Figure 1).

This product is one of the most sensitive endoglycosidase substrates developed. Currently, this substrate has been successfully utilized in mouse and human melanoma heparanase assays. The same type of derivative is also called Reacti-Gel (NW
-65F) (Pierce).

Rockford, I
It is made using L). However, Affigel (A
ffi-Gel) 15 and Reacti-Gel (React
i-Get) (HW-65F) both use very large particles and they retain a significant amount of high molecular weight material within the gel matrix. This is a problem in quantitative heparanase assays and we therefore use Affi-gels of approximately 1-3 microns in diameter with an exclusion limit of Mr 10,000.
) 701 or 702 (Bio-Ra
By using d)), a more desirable assay substrate was developed.

The special composition operation is as follows. Radioactively labeled HS is reduced with sodium borohydride to create a sugar alcohol at its reducing end. The sugar alcohol is converted to a primary aldehyde by periodate oxidation. The aldehyde group is further coupled via a Schiff base to amino-derivatized beads such as Affi-gel 701 and stabilized by reduction with sodium cyanoborohydride.

Furthermore, the proposed operations, as well as the methods we have developed previously, are considered to be easy to use.

Radiolabeled HS whose amino groups are sulfated or acetylated must be aminated at its reducing end with ammonia under reducing conditions.

Affi-gel 702, N-
Conversion to amino-reactive beads is achieved by derivatization with hydroxysuccinimide or N,N'-carbonyldiimidazole, and the aminated radioactive H3 is coupled to amino-reactive Affi-gel 702. Bolton and Hunter (1)
lunter) reagent, although it is disadvantageous due to potential structural changes, +2J=
The use of label H3 makes the substrate more radioactive. On the other hand, the assay can also be improved by the use of fluorescein-labeled H3 for routine clinical studies, and fluorescein-labeled H3 can be rapidly analyzed for degraded fragments by HPLC equipped with a fluorescence detector. suitable for.

Concentration assays for glycosaminoglycan endoglycosidases, such as heparan sulfate endoglycosidase (heparan sulfate), can also be performed in an immunoassay format using polyclonal or monoclonal antibodies, or both, raised against the endoglycosidase. Preferably, Oldberg et al.
1980, Biochemistry (Biochem, )
Antibodies with relatively little cross-reactivity against other endoglycosidases can be used, such as the platelet endoglycosidase described in J.D., Vol. 19, pp. 5755-5762.

The antibodies may be used in a variety of immunoassay techniques to directly measure endoglycosidase proteins.

The endoglycosidase is produced by Barson
) and Yalow (1968), Clinical Chemistry Actor (cl1n, Chem,
Acta) Vol. 22, p. 51, and Miles et al. in 1976, Analytical Biochemistry (Anal.

Immunoradiometric assay (IRMA) reported in Biochem, Volume 61, pages 209-224.
It can be measured using a +251-labeled antigen or antibody. In addition, the endoglycosidase is
Enzyme immunoassay using competitive binding method or IRMA [similar “sand inch” method and rice dam (Wisdom)]
sdom), 1976, Clinical Chemistry (cl1n, Chem,) Volume 22, 124
alkaline phosphatase conjugated to an antibody or endoglycosidase, reviewed on pages 3-1255;
It can be measured using horse radish peroxidase or other enzymes.

In addition, endoglycosidase is produced by Gerso (Gerso)
n) in the Journal of Clinical Immunoassay (J, Cl1n, immunoassay)
The use of fluorescein or other fluorescent compounds, reviewed in Volume 7, pp. 73-81, by Woodhe.
ad) in 1984, Journal of Clinical Immunoassay (J, Cl1n.

It can also be measured by these methods of detection using chemiluminescence, or other labels, as reviewed in vol. 7, pages 82-89. In all these assays, bound endoglycosidase is separated from unbound endoglycosidase by various techniques. These include - solid-phase immobilization of secondary antibodies, avidin-biotin separation using biotin-labeled antibodies and solid-phase avidin,
These include "double antibody" precipitation, or the use of a solid phase antibody to a hapten, such as fluorescein, coupled to a secondary antibody, or separation by the use of a solid phase "secondary antibody."

(“Double antibody” was first introduced in 1969 by Midley
) etc., Acta Endocrinology (ActaE
ndocrinol,) Volume 63, Special Issue No. 142, 24
Defined as another antibody conjugated with an anti-endoglycosidase antibody, as reported on page 7. ) The solid phase systems mentioned above include polymers such as polystyrene, agarose, sepharose, cellulose, glass beads,
and magnetic particles of cellulose or other polymers. The solid phase can be in the form of beads, large or small, or particles, tubes, plates or other shapes.

U.S. Pat. No. 3,89 to Cizesniak is useful for the present invention.
No. 9,298.

The kit includes at least one, or at least two,
or is partitioned to accommodate at least three or more containers and includes a carrier capable of securely holding the containers. The first container contains a purified anti-glycosaminoglycan endoglycosidase antibody (preferably a monoclonal antibody) in solution or lyophilized form or, if the container is a test tube, covalently bound to its interior. Put it in. The second container contains a second anti-glycosaminoglycan endoglycosidase antibody, also preferably a monoclonal antibody. Separately, another container contains a detectably labeled glycosaminoglycan endoglycosidase antigen. When testing for glycosaminoglycan endoglycosidase antigen in a sample, the sample is added into a first container containing monoclonal antibodies, incubated, and antibodies from a second container are added to form a "sandwich.""make. The antibody in the second container is detectably labeled, eg, with a radioactive label or an enzymatic label. A separate container in the kit contains the appropriate enzyme substrate for carrying out the "Eliza" method. A number of variations or permutations to the various techniques used to detect glycosaminoglycan endoglycosidase antigens are also contemplated for the manufacture of this kit. These are a matter of choice determined by ease of handling and rapidity and efficiency of testing.

Quantitative analysis of glycosaminoglycan endoglycosidase antigens can be performed by reading from a standard curve, as is well known in the art. For this purpose, a number of containers can be included in the kit, each containing a different amount of glycosaminoglycan endoglycosidase.

In another embodiment, the antibody is immobilized on a piece of plastic and contacted with a sample suspected of containing the glycosaminoglycan endoglycosidase antigen.

The plastic piece is then contacted with a solution containing a second enzyme-labeled anti-glycosaminoglycan endoglycosidase antibody. This results in the formation of sand inches on the plastic piece. Finally, the introduction of the plastic piece into a coloring solution (such as a substrate for the enzyme) and the detection of color is a rapid, efficient and inexpensive method for the qualitative and It is also a rough method for quantifying glycan endoglycosidase antigens.

The immunoassay of the invention uses antibodies against heparan sulfate endoglycosidase, which distinguish between various glycosaminoglycan endoglycosidases. The methodology described here is superior to other methods for the detection of glycosaminoglycan endoglycosidases in terms of sensitivity and ease of handling.

Similarly, as mentioned above, it is easy to assemble a kit in which the molecular beacon includes a labeled glycosaminoglycan, preferably immobilized through its amino terminus. Such kits would also have to contain specific binding reagents capable of removing bound glycosaminoglycans or fragments thereof from solution. This specific binding reagent can be preconjugated or may be conjugated by the user of the kit and assay. Preferred binding reagents include proteins, more preferably antibodies, and even more preferably monoclonal antibodies.

The development of specific diagnostic tests for glycosaminoglycan endoglycosidase inoculations has become medically desirable for purposes such as tumor detection. Specific diagnostic tests, such as those described herein, may be developed through the use of monoclonal or polyclonal antibodies that specifically bind to glycosaminoglycan endoglycosidases.

These examples are presented to describe preferred embodiments and uses of the invention and are not intended to limit the invention unless otherwise indicated in the claims.

Material 1≦r glucans and enzymes. Bovine lung heparan sulfate (H3) is produced by N, Di, Ferrante (T
X, Huston, Baylor College of Medicine
icine)). The average Mr was determined to be 34,000 by the sedimentation equilibrium method (Nakajima, Ml et al., 198
4th year Journal of Biological Chemistry (J, Btol.

Chem, ), volume 259, pages 2283-2290,
and Irimura, T* et al., 19
Analytical Biochemistry (Ana) in 1983
l, Bio-chem, ) Volume 130, 461-4
p. 68) Heparin derived from pig mucosal tissue (Mr 11.0
00) is M, 'B.

Matthews, Dr. J., C1fonelli and Roden.
(rL, University of Chicago). Shark cartilage chondroitin 6-sulfate (c6S) was obtained from Miles Scientific
ific) (I L, Naperville
1) and further purified by gel chromatography. The average Mr is, as mentioned previously, (Irimura, T, + et al., 19
Analytical Biochemistry (Ana) in 1983
l.

Biochem, Volume 130, Pages 461-468)
It was measured at 60,000. Heparin and N-acetyl-D-glucosamine from bovine lung and pig intestinal mucosa were obtained from Sigma Chemical Company.
micalCo, ) (Mo, St. Louis (St, L)
Obtained from (Ouis)). monosi-alosyl biante
nnary) complex glycopeptide UB-I-b
(M r 2190) is thyroglobulin (Sigma (
(Sigma)) purified from (Irimu)
ra), T* et al. 1983 Analytical Biochemistry (Anal, Biochem,) 1
30, pp. 461-468). Flavobacterium hepa
Heparitinase derived from M. rinum) (EC, 4, 2, 2, 8) was manufactured by Miles Scientific (Mil
esScientific).

High speed gel permeation chromatography.

High performance gel permeation chromatography was performed as previously described (Irimura).
+1.5 etc. 1983, Analytical Biochemistry (Anal, Biochem, ) 130
Volume, pp. 461-468), Flight Gel (Toyopearl)
) TSKHW-55(S) (Gibstauw, NJ
bbstown).

A high-pressure liquid chromatography system (FL,
Riviera Beach L
DC). A 100 microliter aliquot of the sample solution was delivered to the inlet, and chromatographic elution was carried out with a 0.2 M sodium chloride solution at 55° C. at a rate of 1.0 ml/min (Irimu
ra), 'r, 1st prize, 1983, Analytical
Biochemistry (Anal, Bioc-hem,
) Vol. 130, pp. 461-468). In the analysis of radiolabeled substances, the fractions (0,
6 m1) was collected and 3.0 ml of Liquiscint (Comerville, NJ) was collected.
comerville) + National Diagnostics
s)) and mixed with Beckman
) Counted on a LS 2800 liquid scintillation counter (cA, Irvine,
Beckman, Instrument (Beckman I
nstruments )).

Cellulose acetate electrophoresis. caberetti (cappel)
letti) etc. (cappellet
Analytical biochemistry (Ana Ti) etc.
l.

Glycosaminoglycans were analyzed by cellulose acetate electrophoresis according to the method of Biochem, Vol. 99, pp. 311-315).

Titanium Zip Z
one) cellulose acetate plate (TX, Vermont (
Beaumont), Helena Laboratory (He
1ena Laboratory ) + 6.0 X 7
．． 6 cm), and Cappelletti (c
appeletti) etc. (Cabelletti (cap)
Pelletti) et al., Analytical Biochemistry (Anal, Biochem, ) vol. 99,
311-315), 0.5M pyridine-acetic acid (
pH 5,0) at 70V for 60 minutes. During electrophoresis, buffers and cellulose acetate plates were kept below 4°C using ice-cold petroleum ether.

N-desulfation and acetylation of H3. N-desulfation of H3 was performed by Nagasawa and Inouni.
noue ) (Nagasawa etc. 19
1977, Methods in Carbohydrate Chemistry
Chem, Vol. 8, pp. 291-294). Purified sodium salt of H3 was added to AG50WX8
(H type, CA, Richmond
d), converted to pyridinium salt by cation exchange chromatography using a Bio-Rad column.

Fully and partially N-sulfuric acid of H3 was subjected to Hs bipyridinium salt solvolysis in 95% aqueous dimethyl sulfoxide (DMSO) at 50°C for 120 minutes and 20°C for 60 minutes, respectively. The pH of the reaction mixture is adjusted to 9.0 by adding 0.1 M sodium hydride, and the mixture is further dialyzed in running water overnight and against distilled water for 20 hours. N-acetylation of N-desulfated H3 was performed at pHs containing 4% acetic anhydride and 15% methanol.
, o in 4M sodium acetate at 4°C for 3 hours. The reaction mixture was dialyzed overnight in running water and once again against distilled water, then the mixture was lyophilized.

Radiolabeling of H3. To study the effect of chemical modification of H3 on receptivity to melanoma heparanase,
H3 was previously mentioned (Nakajima
) + M, et al., 1984, Journal of Biological Chemistry (J, Biol, Chem
The reducing end was labeled with tritium as described in 259, pp. 2283-2290).

Milligrams of purified H3I was added to a 0.1 M boron buffer 7
NaB (3H) 4 of ZmCi in (+)H8,O)
(340mci/mmol; M A + Boston + - New England Nuclear
)) for 5 hours at 25°C.

After acidification to pH 5 with acetic acid, the reaction mixture was adjusted to pH 5.
Sephacryl S-200 equilibrated with 2M pyridine acetate buffer (pH 5,0)
Chromatography was performed using a column (1, OX 105 cm). A 3H-labeled HS fraction with a special Mr was collected,
Lyophilized. To synthesize radiolabeled H3 for solid-phase heparanase substrates, partially N-desulfated H3
was N-acetylated with (1-”C) acetic anhydride. Part N
- 15 milligrams of desulfurized H3 was added to 4M sodium acetate (
0.15 mC1 of (1-+4C) acetic anhydride (10, OmCi/mmol.

New England Nuclear
-1 and Nuclear)) at 4°C for 4 hours. Furthermore, in the same buffer, in 4% acetic anhydride,
Incubate at 4°C for 14 hours. The reaction mixture was extensively dialyzed against distilled water and lyophilized. Partial N-sulfuric acid, N-(14C)acetylated H3 (PNDS-N
The high Mr fraction of ("C)Ac-H3) was obtained by gel chromatography on a Sephacryl S-200 column as described above.

Reductive amination of IC-labeled H3 and binding to amino-reactive agarose beads. The reducing terminal saccharide of '4C-labeled H3 was reductively aminated as follows. PNDS
-N(14C)Ac-H3 (5 mg) was dissolved in 5 ml of distilled water, mixed with 5 ml of 4 M ammonium formate and 0.8 M sodium cyanoborohydride in methanol, and incubated for 7 days at 50'C. do. The reaction mixture is dialysed against distilled water and lyophilized. The reductive amination product was added to 10 ml of 0. Affi-ge was prepared from the original suspension by dissolving in IN sodium bicarbonate (pH 8,5) and washing with 2-propanol and then water-cooled distilled water.
l) 15 (or Affi-Gel)
10. Mix with Bio-Rad). The mixture is incubated at 4° C. for 24 hours with gentle mixing. Furthermore, the pH was adjusted to 0. I
Adjust to 8.5 with N sodium bicarbonate and continue incubation. After 24 hours, remove 1 ml of the unreacted area on Affi-Gel 15.
1M glycine ethyl ester (pH 8.0) and the beads are incubated again for 6 hours at 4°C. After the reaction is completed, the combined product is 1.5
Thoroughly wash with IJum (M chloride), add 0.1 M sodium acetate, 0.15 M sodium chloride, 0.2% Triton X-100, and 0.05%
Incubate overnight at 37° C. with sodium azide (pH 6.0). The product is further washed with the same buffer and kept at 4°C.

A summary of the above synthetic operations is shown in FIG.

Chemical deacetylation and radioactive reacetylation of heparan sulfate and binding to agarose beads.

14C- or 3H labeled H3 was prepared by chemical deacetylation and radioactive reacetylation as shown below.

9 milligrams of bovine lung [tH3 (Tx.

Houston Baylor College of Medicine (Houston)
Bayloy COColle of Medicine
e), N, Di Ferran
te ) is dried with 1 milligram of hydrazine sulfate over phosphorus pentoxide under reduced pressure at 50° C. for 48 hours. The dry H3 was treated with anhydrous hydrazine (If, Rockford, Pierce Chemical),
0.5 mg), and the mixture was heated at 100° C. for 1 hour in a screw-top tube under a nitrogen cloud. After the reaction, the hydrazine is removed by repeated azeotropy with toluene over a sulfuric acid desiccant under reduced pressure.

To separate the deacetylated H3 from the remaining reagents and partial decomposition products, the completely dried residual aroma was heated to 0.5%.
Dissolve in 5 ml of water and add biogel (BioGe).
l) 0.8X30cm of P-10 (400 meters)
Pass through a gel filtrate on an O column, eluting with distilled water. The void volume fraction is collected and lyophilized. The yield was approximately 60% by weight.

Furthermore, N-acetylated H3 was added to 50 μCi of (14C)-acetic anhydride (
Ma, Boston, NEN, 10
N-acetylation is carried out with 5 mC1 of [3H]-acetic anhydride (400 mci/mmole, NEN) for 18 hours. Complete N-acetylation is achieved by mixing with 0.1 ml of unlabeled acetic anhydride for 1 hour. As above, IC or 3H-labeled H8 was added to BioGel (BioGel) P-10
Purify using a column.

3H-H3 at 0.4M for solid-phase heparanase assay
The reducing end is aminated with a 50% methanol solution of sodium cyanoborohydride at 50° C. for 6 days. Aminated 'H-H3 was purified by gel filtration in the same manner as described above, and the resulting solution was made into a 0.1 M sodium carbonate solution. lQ'cpm aminated 'H-H3, 1.0 m
j! Affi-Gel L 5,
It was added after washing with Improper Tool and cooling water according to the manufacturer's recommendations. The coupling reaction was continued for 48 hours at 4° C. with stirring. Furthermore, the agarose beads were washed repeatedly with 4M sodium chloride to remove non-covalently attached 'H-H3 from the beads.

Manomena cells and cell culture. In this study, we investigated the metastatic murine B16 melanoma subline (B1
6-FIO) and 14 established cell lines of human malignant melanoma were used. The human melanoma cells used were SK-MEL-19, SK-MEL-23, and SK-MEL-19.
MEL 93 (DX 1), SK-MEL-9
3 (DX3), SK-MEL-93 (DX6), H
s 294T.

Hs852T, H3939, T294, M2O, RON
, BMCL, A375 Parent, and A735 Me
t Mix +. A375 Met Mix cells are A375 Met Mix cells in athymic nude mice.
Prepared from natural lung metastases of 375 parental cells, both A3
75 cell lineages are I, J, Fiddler
r) Received from Dr. (Tx, Hyuston (H)
ous ton) + (University of Texas-M, D, Anderson Hospital and Tumer Institute (The University
y ofTexas M, D, Anderson
Ho5pital and TumorInstit
a Melanoma cells were placed in 95% air-5% C
10% fetal bovine serum CUT under humidified conditions of O7 atmosphere,
Logan, Hyclone Sterile System Inc.
e+ 5terile+ 5ysteIIl, Tnc
+ ) added and antibiotic-free, Dulbeco (Dul
becco) and the modified minimal basal medium of Ham (Ham)
F12 medium (Grand Island, NY)
nd l5land) Gibco Laboratory (Gib
co Laboratories), DMEM/F12
) were grown in plastic tissue culture dishes containing a 1:1 mixture of All cell cultures were grown using a Mycoplasma Detection System (MD, Gaithersberg).
burg), Bethesda.

Research Laboratories, (Bethesda R
e5archLaboratories ) B
RL Mycotest (Myc.

test)), it was confirmed that there was no mycoplasma contamination.

Preparation of cell extracts. The collected melanoma cells were
g +″ M B24-free, 2 out of 21 in DPBS
Harvested after 11 days. Single cell suspension 0.14
M Sodium Chloride, 10mM 1-Lys-HCl Buffer (
The cells were washed twice by low-speed centrifugation (pH 7.5), and the viability (usually 95% or more) was checked by exclusion of Trivan blue dye. Cells were transferred to chilled 50mM 1-Lys-HCl buffer (1) H7.5 containing 0.5% Triton X-100.
), suspended at 6x106 cells/mA,
Extraction was carried out at 4°C for 5 minutes and then at 4°C for 1 hour. 4℃,
After centrifugation at 9800×g for 5 minutes, the supernatant was collected. The protein content of the centrifuged extract is determined by the amount of Triton X-1 present in the sample.
To correct for 00, the measurement was carried out by modifying the Lowry method (Nakajima).
M. et al., 1984, Journal of Biological Chemistry (J. Bio. Chem.),
259, pp. 2283-2290).

Enzymatic degradation of modified and unmodified H3. In enzymatic digestion experiments, 3H-labeled H3 substrate (10°Cg) was diluted with 20mM D-
1% i acid 1.4-lactone (S A L.

0.15 M sodium chloride, 0.2% Triton
200 μl containing 0.00 and 0.05% sodium azide
816-FIO cell extracts (80 °C) in 0.1 M sodium acetate buffer (pH 6.0) (reaction buffer A).
g protein) and incubated. Incubation was carried out at 37°C with slow agitation.
The mixture was cooled to 4°C and the reaction was stopped by adding 20 μl of Trif00 acetic acid to a final concentration of 5%.

A supernatant was obtained by centrifugation at 980QXg for 5 minutes, and analyzed by high-speed gel permeation chromatography.

Heparanase assay using solid phase substrate. Affigel (A
ffi-get) PNDS-N (
"C) A suspension of Ac-B3 was mixed with melanoma cell extract and reaction buffer A4 containing 20mM 5AL was added."
00μ! incubated inside. The enzymatic reaction was stopped by cooling the solution to 4° C. and adding an additional 40 μl of 50% trichloroacetic acid. After incubation for 10 minutes at 4°C, the mixture was incubated at 9800×g for 5 minutes.
It was centrifuged for a minute and the supernatant was collected. 200μ of the supernatant
1 aliquot of 55 μl. ON Sodium Hydroxide and 4ml Liquis-cint
(National Diagnostics)
1 Diagnostic) and counted in a Beckman LS 2800 liquid scintillation counter.

/-7/- B in H3 degradation by maheparanase
Effect of N-desulfation and N-acetylation on 3.

In order to label purified B3 with radioactive molecules without using coupling reagents that would cause significant changes in the H3 molecular structure, we replaced some N-sulfate groups with N (14c
) Substituted with an acetyl group. The idea is that B16 melanoma heparanase is inactive against heparin but highly active against various H3 molecules, and that B3 has a high content of N-acetyl and a low content of N-sulfate. This is based on our previous observation that it differs from heparin in this respect.

The effect of N-desulfation and N-acetylation of B3 on melanoma heparanase acceptability was assayed using B3 whose reducing terminal sugar was labeled with tritium. B3 purified from bovine lung has N-acetyl-D-glucosamine at its reducing end (Nakajima).
Nakajima) + M11 et al. (1984), Journal of Biological Chemistry (J,
Biol, Chem, ) 259, 2283-22
90), H8 was reduced with NaB("H)a. HS whose reducing end was labeled with tritium was treated with DMSO
The N-desulfation (3H) B3 was further N-acetylated with acetic anhydride. These three 3H-labeled, chemically modified H3 molecules were buffered in 0.5 M pyridine-acetate buffer (pH 5,0
) was analyzed by cellulose acetate electrophoresis. B3. under the electrophoretic conditions described in Materials and Methods. N-desulfation H
3 (NDS-B3) and N-desulfated N-acetylated H3
The relative mobilities of (NDS-NAc-B3) are 3.
They were 30, 2.55 and 2.90. These findings indicate that N-desulfation of B3 results in a significant loss of negative charge, but that the total charge is partially regained by acetylation of the free amino acid. .

The average molecular sizes of NDS-B3 and NDS-NAc-B3 were determined by high performance gel permeation chromatography and were found to be 30.000 and 31.500, respectively. (Figure 2) , B3, NDS-B3
and NDS-NAc-B3 were each incubated with B16 melanoma cell extract in the presence of SAL (a potent exobetadalcuronidase inhibitor) and the incubation products were analyzed by high performance gel permini-silon chromatography. All these substrates were cleaved at high rates by melanoma heparanase, and although the Mr of the resulting fragments was characteristic of each fragment, the elution profiles of their degradation products were the same. (
Figure 2) Degradation of each chemically modified HS was generally inhibited by the addition of heparin purified from bovine lung or porcine intestinal mucosa.

(data not shown). The result is that N- in H3
Sulfuric acid is not important for melanoma heparanase recognition or cleavage, indicating that modification of the sulfated amino group in H3 does not significantly affect degradation by heparanase.

N-desulfated and N-acetylated heparin. The known structures of HS and heparin indicate that N-desulfation and subsequent N-acetylation of heparin creates local structures similar to those present in H3. Heparin is a potent inhibitor of B16 melanoma heparanase (Nakajima (N
aKajima), M.

(1984), Journal of Biological Chemistry (J, Biol, Chew, ),
259, pp. 2283-2290). However, its heparanase inhibitory activity is lost upon removal of N-sulfate (Irimura et al., 1985, Journal of Cellular Biochemistry (J.
Ce11. Biochem, ) 9 A, p. 148).

Since the above results indicate that N-sulfate in H3 is unnecessary for cleavage by melanoma heparanase,
N-desulfated N-acetylated heparin was used as heparanase substrate. Pig intestinal mucosa-derived heparin (Mr.
11,000) (Nakajima)
, M. et al., 1984, Journal of Biological Chemistry (J, Biol.

Chem, Volume 259, Pages 2283-2290)
was N-desulfated and N-acetylated by the procedure used in the preparation of NDS-NAc-H3. The product, N
-Desulfated N-acetylated heparin (NDS-NAc-heparin) had an apparent Mr value of approximately 10,500 as determined by high performance gel permeation chromatography. Then, 0.2M pyridine-acetic acid buffer (
The relative electrophoretic mobility in cellulose acetate at pH 5, 0) is 0.0 when 3H-labeled heparin is 1.00.
It was 87. 3H-labeled heparin and 3H-labeled NDS
-NAc-heparin was incubated with 816 cell extracts and the reaction products were analyzed by high performance gel permeation chromatography. The degradation rate of the original substrate is
Previously reported (Nakajima)
+ M + et al., 1984, Journal of Biological Chemistry (J, Biol.

Chem, Volume 259, Pages 2283-2290)
Calculated from the decrease in the high Mr fraction area by half of the substrate peak. First 6 incubations with B16 cell extract
In time, less than 5% of heparin was degraded, while NAc-
Approximately 20% of the heparin was fragmented.

However, NDS-NAc-heparin was not cleaved, and furthermore, even after long incubation, there was no shift of the main peak of NDS-NAc-heparin to lower Mr in high performance gel permeation chromatography. . This indicates that N-desulfation and subsequent N-acetylation of heparin may result in the creation or exposure of a heparanase-accepting glucuronosyl bond on a portion of the heparin molecule. In this way, NDS-NAc
- Heparin cannot be used as a substrate for melanoma heparanase assays.

Synthesis of solid phase substrates for melanoma heparanase assays. The procedure for the synthesis of solid phase substrates for heparanase is illustrated in FIG. To minimize the effect of the radiolabel on the H3 structure, H3 was prepared in 95% aqueous DMS solution.
Only partial desulfation was performed by solvolysis at 0°C for 1 hour. Partially desulfated H3 was acetylated with (1-14C) acetic anhydride as described in Materials and Methods. The remaining free amino groups were completely acetylated with acetic anhydride. In these steps, PNDS-N (“C:l Ac-H3(M
r 33,000) was obtained. The relative mobility of PNDS-N ("C)Ac-H5 in cellulose acetate electrophoresis is 3.15, which means that PNDS=N ("C)Ac-
As expected, the total charge of ll5 is NDS-NAc-H3
This shows that it is closer to that of unmodified HSO than that of .

The reducing terminal saccharide of PNDS-N ('4C)Ac-H3 was reductively aminated with 2M ammonium formate and 0.4M sodium cyanoborohydride in 50% methanol. The product, which has a free amino group only at its reducing end, was added to the Ara 4' gel (Affi-get
) 10 or Affi-gel
15 was coupled to amino-reactive agarose beads. Affi gel backed to 0.5mj2
gel) 15 and 5 mg of PNDS-N (I4C)
, upon incubation of Ac-H3, PNDS-N (
"C) Ac-H3 is 10.2% immobilization gel 0.5
0.51 mg of PNDS-N C'4C per m1t)
Ac-B3), 0.5 ml Affi-Gel (A
ffi-gel) 15 to 10 mg PN
Even if the concentration of DS-N (”C)Ac-B3 is increased,
The conditions used did not significantly affect binding efficiency.

PNDS-N (14C)Ac-B3 was added to Affigel (
＾ffi-get) Affi-gel under the same conditions used when binding to 15.
It was also combined with 10. However, between pH 7.5 and 8.5, the binding efficiency was as low as 1% or less. Therefore, the positively charged spacer in the amino-reactive site of Affi-gel 15
i-gel) PNDS-N (1C)Ac-B
It may be important for the efficient combination of 3. One of the best heparanase assay substrates was produced using Affi-gel 15. It was immobilized on agarose with only one covalent bond at its reducing end. PNDS-N (I4C)Ac-B3.

immobilized on agarose beads Enzymatic degradation of PNDS-N (I4C) Ac-B3.

PNDS-N immobilized on agarose gel (”C)
The sensitivity of Ac-B5 to HS degrading enzyme was determined using 0.1 M sodium acetate buffer containing 1 mM calcium acetate.
(linker (Linker
) + et al., 1972 Methods Enzymol, Volume 28, 9
02-911 pages).

PNDS-N immobilized on agarose showed that most of the 14C activity (82%) appeared in the supernatant of the incubation mixture after 24 h of incubation.
(I4C)Ac-B3 was shown to be highly susceptible to the action of HS degrading enzymes.

Even if incubation continues, the remaining PND
SN (14C)Ac-B3 was not released from the gel. This is Flavobacterium (Flavobacterium)
A. acterium) is explained by the limitations of using heparitinase. Same amount of substrate (4500cpIll)
B16 for various times in the presence of 20mM 5AL to prevent subsequent degradation by exoglycosidases.
and incubated with cell extract. The relationship between the amount of cell extract added (μg protein) and the released I40 activity is shown in FIG. In this case, the maximum amount of Inc activity released was 56% of the total I4C activity present in the solid phase substrate. Some 14C activity was not released by melanoma heparanase because incubation of B3 or chemically modified B3 with B16 cell extracts resulted in large fragments with native reducing ends. (Figure 2). A proportional relationship between the amount of cell extract added and the +40 activity released was found at each incubation time (Figure 3).

Since the results of the 12-hour incubation have a linear relationship over the widest range of cell extract amounts, we
) The decomposition of Ac-B3 was measured.

The effect of heparin on the degradation of solid phase substrates was
An equal amount of substrate (15 parts g
) was studied by adding heparin derived from pig intestinal mucosa or heparin derived from bovine lung. Each heparin addition corresponds to our previous results (Nakajima
a), Mo et al., 1984, Journal of
Biological Chemistry (J, Biol, C
Hem., Vol. 259, pp. 2283-2290), and the inhibition of the degradation of the solid phase substrate occurred by up to 80%.

Measurement of heparinase activity in human melanoma cells by using PNDS-N (''C)Ac-B5 immobilized on agarose beads. Using PNDS'-N (''C)Ac-B3 immobilized on agarose beads. , next 1
Five human melanoma cell lines were tested for heparinase activity. SK-MEL-19, SK-MEL-23
, SK-MEL-93 (DXl), SK-MEL-
93 (DX3), SK-MEL-93 (DX6)
, Hs294T, Hs852T.

Hs939, T294, M40. RON, BMCL.

A375 Parent, A375 Met Mix
and A375 M6° As shown in Table 1, in the presence of SAL, all human melanoma cells showed the ability to degrade H3. Among these human malignant melanoma cell lines, SK-MEL-93 (DXI), SK-MEL-
93 (DX6), Hs 939, M40. A375
Met Mix, and six species of A375 M6 have a larger H than the mouse 816 melanoma sublineage FIO.
3 resolution was significantly demonstrated.

Interestingly, A375 M6 cells are associated with Aciminc (
athymic) were selected from the A375 parental cells due to their ability to metastasize and grow in the lungs of nude mice.

They have very high metastatic potential whereas A375 parental cells have very low metastatic potential.

(Kozlowski et al., 198
4th year, Journal of National Cancer Institute (J, Natl, Cancer
In5t, ) Vol. 72, pp. 913-917). Therefore, the hepananase activity of A375 cells may be correlated with their natural lung metastasis potential.

We previously reported that B16 melanoma cells themselves or
B16 with high lung metastasis and proliferation! [Extracts of the cell sublineage I degrade purified HS at a higher rate than B16 cells with low lung metastatic potential (Nakajima et al., 1983, Science 220, 611-613) page), and that B16 melanoma HS-degrading endoglycosidase is an endo-beta glucuronidase (heparanase) (Nakajima (
Nakaj 1IIIa) + M, et al., 1984, Journal of Biological Chemistry〇, Biol, Chem, ) vol. 259, 2283
~2290 pages).

Heparan sulfate degrading activity in human malignant melanoma cells Melanoma cell line Heparan sulfate degrading activity 1° standard deviation above mean value (cpIII) a, Heparanase assay was performed using Triton
-100 cell extracts (2,4 x 10' cells) were immobilized on PNDS-N (14C)Ac-H3 on agarose beads for 12 hours at 37°C.
This was done by incubating for hours.

Experimental details are described in the Materials and Methods section.

In the presence of heat-inactivated enzyme, the radioactivity released is subtracted from the raw data.

b, A375 Met Mix and A375 M6 cells derived from lung metastasis of A375 parental cells in asmic nude mice have very high sudden lung metastasis potential, whereas A375 cells have very low sudden lung metastasis potential. Not yet.

Heparanase activity in serum from tumor-bearing hosts.

Preparation of serum. Serum was drawn by static swine puncture without anticoagulant and allowed to clot for 1 hour at 22°C. 800 samples of that
Centrifugation was performed at 4°C for 10 minutes at X9, and further centrifuged at 1600Xg for 15 minutes. The resulting serum was divided into small aliquots, snap frozen in liquid nitrogen, and stored at -80°C until analysis.

Blood heparanase.

The serum was adjusted to 0.1% containing 0.15M sodium chloride.
Diluted 5 times with M sodium acetate buffer (pl+6.0). A 200 microlitre aliquot of the diluted serum was added in the same buffer to 200 μl of the radiolabeled solid phase substrate suspension (3,000 cpm).

10Mg) and incubated at 37°C. At various incubation times, the enzyme reaction was stopped by cooling to 4° C. and centrifuged at 9800×g for 5 minutes. A 200 microliter aliquot of the supernatant was extracted and 1.
Neutralize with 55 μl of ON sodium hydride and add 4
ml! Liquiscint
(National
Diaqnostic). The radioactivity was measured on a Beckman LS 2800 liquid scintillation counter. There was a proportional relationship between incubation time and enzyme reaction. The activity was reported in units per milliliter of serum. Activity 1
A unit represents the amount of enzyme that releases 1 Mg of H3 per minute.

20 melanoma patients at various stages of the disease and 1
Sera from five normal adults were assayed for heparanase activity, and the results are shown in FIG. The mean value and standard deviation of heparanase activity in serum from melanoma patients and normal adults were 0.0177 ± 0.00, respectively.
75, and 0.0096±0.0025 U/ml. The serum of some patients treated with chemical therapy showed normal heparanase activity levels.

Very metastatic mammalian adenocarcinoma M
TL, 3 cells (1x 106 cells) were transferred to F3 cells of suitable age females.
44 Rat left inguinal m.
subcutaneously injected into the amary fat pad).

After injection, the rats were sacrificed at various times and the tumor size, number of lung metastases and their serum heparanase activity were measured. Heparan sulfate-degrading activity in serum increased with time after subcutaneous injection of MTLfi3 cells (Fig. 5). The activity in the serum was correlated with the size of the second tumor (correlation coefficient r=0.110. Figure 6). Sera from rats with many metastases in the lymph nodes and lungs showed much higher heparan sulfate activity compared to sera from rats with fewer or no metastases.

Melanoma heparan sulfate 1° melanoma cells (mouse B16 melanoma subline B16
-BL6 or human melanoma Hs939 cells),
DME/F supplemented with 5% heat-inactivated fetal bovine serum
Cultured in a 1:1 mixture of 12 medium. The assembled cells were harvested by treatment for 10 minutes in PBS containing 2a+M EDTA and 0.14M NaCl, 10m
M) Washed twice with lithium-hydrochloric acid buffer (pH 7.2). The following steps are performed at 4°C. Cells (2 x 10a) were incubated with 0.2% Triton X-10.
30 ml of 11011I Tris-HCl buffer (pH 7,2) containing 0,10 MMPMsF (Buffer A)
Extract for 1 hour.

After centrifugation at 30.0OOX g for 30 minutes, the supernatant (approximately 1.5
mg protein/mj was collected and loaded onto a Conhanavalin A-Sepharose 4B column (2
X10cm) and charge it to 10n+j! buffer A of
After washing with water, the adsorbed substance was eluted with a 1M alpha methyl-D-mannoside solution in buffer A.

The eluate was passed through a heparin-Seph 10-S CL-6B column (2 x 10 cm) equilibrated with 0.15 M sodium chloride, 50 mM Lys-HCl buffer (pH 7.2) containing 0.2% Triton X-100. The column was washed with 100 mj' of the same buffer.
and 0.15 M sodium chloride, 50 mM Tris-
After washing with 100 ml of hydrochloric acid buff 77 (pH 7.2), the heparin-binding protein was further eluted with a linear salt gradient (0.15M to 1.2M sodium chloride). Collect the heparanase active fraction and add 0.15M sodium chloride and 0.01M potassium phosphate (pH 6.0).
Dialyzed with 50. After centrifugation in OOOXg for 30 minutes,
The supernatant was charged onto a hydroxyapatite column (1.5×45 cm) equilibrated with 0.15 M sodium chloride and 0.01 M potassium phosphate (pH 6.0). Heparan sulfate was mixed with 0.15 M sodium chloride (pl+6
．． Potassium phosphate linear concentration gradient (0, OI
It was eluted from M to 0.6 M). YM heparanase fraction
It was concentrated by ultrafiltration using a -10 membrane, and further purified by Sepharose CL-6B gel filtration. Sepharose CL-6B chromatography was carried out at 0.
15M sodium chloride and 20mM potassium phosphate (p
H6,0). Next, chromatography using Sepharose G-150, 0.5M sodium chloride,
The test was carried out using 25mM Tris-HCl (pH 7.5). The single heparanase obtained from this Sepharose chromatography contained glycoproteins with M r 96.000.

Human melanoma heparanase.

Melanoma heparanase is active between pus, s and 7.5 and degrades heparan sulfate, but not other glycosaminoglycans. Heparin and dextran sulfate are potent inhibitors of melanoma heparanase.

Husband-1--1 Heparan sulfate (JIS) + -
N, 0-desulfated HS + -N
, 0-desulfation, N-acetylated H5+
-N-desulfation, Its +
-N-desulfation, N-acetylation H3+
-Heparin-
++N, 0-sulfated heparin --N, 0-desulfated, N-acetylated heparin ±
-N-desulfation
--N-desulfation, N-acetylated heparin ± 10N-m sulfur M,
N-methylated heparin − =
Lupoxyl reduction heparin
1.3H-labeled glycosaminoglycan was mixed with 20mM D-sugar acid 1,4-lactose (
SAL) in the presence of 0.15M NaCl2, 0.2%
Triton X-100 and 0.05%N
a 0.1 M sodium acetate buffer containing N3
(pH 6,0) at 37°C for 6 hours.
0 Mg protein). The decomposition rate is
Determined by measuring the decrease in the area of the glycosaminoglycan beak half of the high Mr value (see Figure 2) ÷: 80%
or more, ±: 5% to 15%, -: 5% or more (standard deviation 5.
0% or less).

2.5 micrograms of unlabeled glycosaminoglycans were added to the incubation mixture of 3H-labeled H3 from bovine lung and cell extract.

Inhibition of H3 degradation was determined by measuring the decrease in the area of the HS peak at half the high Mr value.

++: 80% or more inhibition, +: 25-50% inhibition, ±: 5
~25% inhibition, -: 5% or less inhibition (standard deviation 5% or less)
.

Changes may be made in the arrangement, operation and arrangement of various parts, elements, steps and procedures described herein without departing from the concept and scope of the invention as defined in the claims.

[Brief explanation of drawings]

Figure 1. Synthesis of solid phase heparanase substrate; chemical modification and radiolabeling of H8 and its coupling to amino-reactive agarose gel beads. FIG. 2: High performance gel permeation chromatography elution curves of unmodified and chemically modified H3 before and after treatment with B16 melanoma heparanase. H3, heparan sulfate, NDS-H3, N-desulfated heparan sulfate; NDS-
NAc-H3, N-desulfated, N-acetylated heparan sulfate. These glycans labeled with tritium at their reducing ends and their fragments generated by incubation with 816 melanoma cell extract in the presence of SAL were gelated at a flow rate of 1/1 yle at 55°C. Fractogel)-TSK
Chromatography was carried out by flowing 0.2 M sodium chloride through two HW-55(S) (0.7 x 75 cm) columns connected in series. Arrows (a) to (e) indicate the elution positions of standard glycans: (a) C6C in shark cartilage;
65 (60,000), (bl Bovine Lung HS (M
r 34,000), (c) Pig mucosal tissue (M r
11,000), (d) Monosialosyl biantennary derived from pig thyroglobulin (@H□5ialosyl
biantennary) complex glycopeptide (M
r 2190), (el N-acetyl-D-glucosamine (Mr 221).
Partially N-desulfated N immobilized on agarose
(''C) Acetylated heparan sulfate (PNDS-N (
"C) Dose-dependent degradation of Ac-HS). PNDS-N immobilized on agarose (C) Ac-HS (4500
cpm) with various amounts of 816 cell extract for 6 hours ().
, 12 hours (0), and 24 hours (), or inactivated (100° C., 5 minutes) at various amounts of B1.
6 cell extract in the presence of SAL for 12 hours (). The radioactivity released from half volume of supernatant was plotted against the amount of cell extract added (μg protein). FIG. 4 shows heparanase activity levels in the serum of patients with malignant melanoma and normal individuals. Figure 5 shows heparanase activity levels in the serum of rats injected with highly metastatic athenocarcinoma. FIG. 6 shows the relationship between rat serum heparanase activity and the size of secondary metastatic tumors. FIG. 7 shows the relationship between rat serum heparanase levels and the number of metastases derived from malignant tumors. Figure 8 shows the substrate hydrolysis positions for melanoma heparan sulfate. J/6-710 cell extract (1,,1g evening) bacteria) normal adult melanoma patient

Claims (72)

[Claims] (1) A method for producing a solid phase substrate that, when hydrolyzed by a glycosaminoglycan endoglycosidase, yields a soluble radioisotope-labeled product, the method comprising: (a) converting a large amount of glycosaminoglycans at least in part to N-
(b) radiolabeling the at least partially N-desulfated or N-deacetylated glycosaminoglycans with a radioisotope-labeled acyl anhydride or acyl halide; (c) completely N-acylating the radioisotope-labeled glycosaminoglycan with an acyl anhydride or acyl halide; (d) said radioisotope. reductively aminating the reducing end of the labeled and N-acylated glycosaminoglycan to produce a radioisotope-labeled amine-terminated glycosaminoglycan; (e) a radioisotope-labeled amine; attaching a terminal glycosaminoglycan via its terminal amine to an amine-reactive solid support to form the solid substrate. (2) A method for producing a solid phase substrate that, when hydrolyzed by heparan sulfate endoglycosidase, yields a soluble radioisotope-labeled product, the method comprising: (a) converting a large amount of heparan sulfate at least in part to N-desulfation or N- (b) radiolabeling the at least partially N-desulfated or N-deacetylated heparan sulfate with radioisotope-labeled acetic anhydride to produce radiolabeled heparan sulfate; c) completely N-acylating the radioisotope-labeled heparan sulfate with an acyl anhydride or acyl halide; (d) reductively aminating the reducing end of the radioisotope-labeled heparan sulfate; (e) coupling the radioisotope-labeled amine-terminated heparan sulfate via its terminal amine to an amino-reactive solid support to form the solid substrate; The method includes the steps of: (3) The method of claim 1 or 2, wherein at least a portion of the N-desulfation comprises solvolysis in dimethyl sulfoxide. (4) Claims (1) or (2), wherein at least some of the N-deacetylation includes hydrazinolysis.
The method described in section. (5) the radioisotope-labeled acyl anhydride or acyl halide contains a ^1^4C or ^3H atom;
A method according to claim (1) or (2). (6) Claims (1) or (2) wherein the radiolabeling step is carried out with ^3H or ^1^4C acetic anhydride.
2) Method described in section 2). (7) The method of claim (1) or (2), wherein the reductive amination step includes the use of an ammonium salt and sodium cyanoborohydride. (8) further comprising (f) blocking unbound amine-reactive sites on the solid support by aqueous incubation with an alkali metal borohydride or cyanoborohydride and a water-soluble amine-containing compound. A method according to claim (1) or (2). (9) further comprising: (f) blocking unbound amine-reactive sites on the solid support by aqueous incubation with sodium cyanoborohydride and ethanolamine or glycine ethyl ester. The method according to scope item (1) or item (2). (10) The method according to claim (1), wherein the glycosaminoglycan is hyaluronic acid, chondroitin 4-sulfate, chondroitin-6 sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or a combination thereof. (11) A solid phase substrate that, when hydrolyzed by a glycosaminoglycan endoglycosidase, yields a soluble radioisotope-labeled product, the solid phase substrate comprising:
(b) radiolabeling the at least partially N-deacylated or N-desulfated glycosaminoglycan with a radioisotope-labeled acyl anhydride or acyl halide; (c) completely N-acylating the radioisotope-labeled glycosaminoglycan with an acyl anhydride or acyl halide; (d) said radioisotope. (e) reductively aminating the reducing end of the labeled glycosaminoglycan to produce a radiolabeled amine-terminated glycosaminoglycan; coupling to an amino-reactive solid phase support with an amine to form the solid phase substrate. (12) a solid phase substrate that produces a soluble radioisotope-labeled product when hydrolyzed by heparan sulfate endoglycosidase, comprising: (a) at least partially N-desulfating a large amount of heparan sulfate; (b) a small amount of heparan sulfate; (c) producing radiolabeled heparan sulfate by radiolabeling partially N-desulfated heparan sulfate with radioisotope-labeled acetic anhydride or acetyl halide; (c) converting the radioisotope-labeled heparan sulfate into acyl anhydride; (d) reductively aminating the reducing end of the radioisotope-labeled heparan sulfate to produce radioisotope-labeled amine-terminated heparan sulfate; (e) attaching a radioisotope-labeled amine-terminated heparan sulfate via its terminal amine to an amine-reactive solid phase support to form the solid phase substrate. (13) further comprising (f) blocking unbound amine-reactive sites on the solid support by aqueous incubation with ethanolamine or glycine ethyl ester and sodium cyanoborohydride. Clause (11) or (12)
Solid phase substrate as described in section. (14) The solid phase substrate according to claim (11), wherein the glycosaminoglycan is hyaluronic acid, chondroitin 4-sulfate, chondroitin-6 sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or a combination thereof. . (15) A solid phase substrate that produces a soluble radioisotope-labeled product when hydrolyzed by a glycosaminoglycan endoglycosidase, the substrate being a radiolabeled N
- a glycosaminoglycan having an acyl group, wherein said glycosaminoglycan is reductively aminated at its reducing end to produce an amine end, and said glycosaminoglycan is bound to an amine-reactive solid matrix by said amine end; solid phase substrate. (16) The solid phase substrate according to claim (15), wherein the acyl group is an acetyl group. (17) The labeled N-acyl group is ^3H or ^1^4
The solid phase substrate according to claim (15), which comprises C. (18) the glycosaminoglycan is heparan sulfate,
The endoglycosidase is heparanase, patent No.
15) The solid phase substrate described in section 15). (19) The solid phase substrate according to claim (15), wherein the amine-reactive solid matrix comprises agarose beads. (20) Claim No. 15, wherein the amine-reactive solid matrix comprises cyanogen bromide-activated agarose beads.
) The solid phase substrate described in section 2. (21) The solid phase substrate according to claim (15), wherein the amine-reactive solid matrix comprises N-hydroxysuccinide ester of agarose. (22) The solid phase substrate of claim (15), wherein the amine-reactive solid matrix comprises periodate-activated agarose. (23) An assay procedure for measuring glycosaminoglycan endoglycosidase enzymatic activity in a biological sample, comprising: (a) adding a portion of the sample to a solid phase with radiolabeled amine-terminated glycosaminoglycans attached simply by the terminal amino group; (b) measuring the soluble radiolabel as a function of glycosaminoglycan endoglycosidase enzyme activity. (24) An assay procedure for measuring heparan sulfate endoglycosidase activity in a biological sample, comprising: (a) introducing a portion of the sample into a buffered aqueous mixture with a radiolabeled amine-terminated heparan sulfate bound to a solid phase simply by the terminal amino group; (b) measuring the soluble radiolabel as a function of heparan sulfate endoglycosidase enzyme activity. (25) A method for detecting the presence of metastatic tumor cells in a patient, comprising: (a) obtaining a serum sample from a control individual and a patient suspected of having metastatic tumor cells; (c) incubation in a buffered aqueous medium comprising a labeled glycosaminoglycan labeled with a radioisotope N-acetyl group, the labeled glycosaminoglycan being bound to the matrix by its terminal amine; measuring the concentration of soluble radiolabeled product formed as a function of time; (d) comparing the concentration of soluble radiolabeled product formed by patient serum and serum from a control individual; metastatic tumors; the patient having the cells develops a significantly higher concentration. (26) A solid phase substrate that, when hydrolyzed by a glycosaminoglycan endoglycosidase, yields a soluble product labeled with a detectable signal, which does not interfere with the hydrolysis of the labeled glycosaminoglycan by the glycosaminoglycan endoglycosidase. A solid phase substrate comprising a glycosaminoglycan with a label attached to a solid matrix by a single covalent bond through its reducing terminal end. (27) A method for producing a solid phase substrate that yields a soluble labeled product when hydrolyzed by a glycosaminoglycan endoglycosidase, the method comprising: (a) converting a large amount of glycosaminoglycans at least in part to N-
(b) labeling the amino groups of the at least partially N-desulfated or N-deacetylated glycosaminoglycans with a label that produces a detectable signal; producing a glycosaminoglycan; (c) completely N-acylating the amino group of the labeled glycosaminoglycan with an acyl anhydride or an acyl halide; (d) the labeling and N-acylation. reductively aminating the reducing end of the labeled glycosaminoglycan to produce a labeled amine-terminated glycosaminoglycan; (e) subjecting the labeled amine-terminated glycosaminoglycan to an amine reaction with the terminal amine; binding to a solid support to produce the solid substrate. (28) A method for producing a solid phase substrate that yields a soluble labeled product when hydrolyzed by heparan sulfate endoglycosidase, the method comprising: (a) at least partially N-desulfating or N-deacetylating a large amount of heparan sulfate; (b) labeling the amino groups of the at least partially desulfated or N-deacetylated heparan sulfate with a label that produces a detectable signal to produce labeled heparan sulfate; (c) labeled heparan sulfate; a step of completely N-acylating the amino group of heparan sulfate with an acyl anhydride or an acyl halide; (d) the labeled amine-terminated heparan sulfate by reductively aminating the reducing end of the labeled heparan sulfate; (e) attaching a labeled amine-terminated heparan sulfate via its terminal amino group to an amino-reactive solid phase support to form said solid phase substrate. (29) Claim No. 27, wherein the labeling step is carried out with a fluorescent label, an enzyme label, or a radioisotope label.
) or the method described in paragraph (28). (30) A solid phase substrate that produces a soluble labeled product when hydrolyzed by a glycosaminoglycan endoglycosidase, the solid phase substrate comprising:
(b) labeling the amino groups of the at least partially N-deacylated or N-desulfated glycosaminoglycans with a substance that produces a detectable signal; producing a glycosaminoglycan; (c) completely N-acylating the amino group of the labeled glycosaminoglycan with an acyl anhydride or an acyl halide; (d) producing the labeled glycosaminoglycan. reductively aminating the reducing end to produce a labeled amine-terminated glycosaminoglycan; (e) attaching the labeled amine-terminated glycosaminoglycan to an amino-reactive solid support via its terminal amino group; A solid phase substrate produced by a method comprising: binding to produce said solid phase substrate. (31) a solid phase substrate that yields a soluble labeled product when hydrolyzed by heparan sulfate endoglycosidase, comprising: (a) at least partially N-deacylating or N-desulfating a quantity of heparan sulfate; b) labeling the amino groups of at least partially N-deacylated or N-desulfated heparan sulfate with a substance that produces a detectable signal to produce labeled heparan sulfate; (c) labeled heparan sulfate; (d) reductively aminating the reducing end of the labeled heparan sulfate to produce a labeled amine-terminated heparan sulfate; (e) attaching a labeled amine-terminated heparan sulfate via its terminal amino group to an amino-reactive solid phase support to produce said solid phase substrate. (32) The solid phase substrate according to claim (30) or (31), wherein the label is a fluorescent label, a radioisotope label, or an enzyme label. (33) a solid phase substrate that, when hydrolyzed by a glycosaminoglycan endoglycosidase, yields a soluble product having a detectable label; A substrate comprising a glycosaminoglycan attached to a solid matrix by a terminal hexose. (34) A method for producing a liquid phase substrate for use in assaying human glycosaminoglycan endoglycosidase, which is a substrate that produces a labeled product when hydrolyzed by glycosaminoglycan endoglycosidase, the method comprising: (a) glycosaminoglycan endoglycosidase; (b) tagging an unlabeled site of the labeled glycosaminoglycan with a molecule; (c) labeling and tagging the labeled glycosaminoglycan at one or more sites with a label that produces a detectable signal; (d) incubating the glycosaminoglycan in a buffered aqueous solution with a human biological sample suspected of containing glycosaminoglycan endoglycosidase; (d) labeling and tagging the glycosaminoglycan endoglycosidase of the glycosaminoglycan substrate; separating the labeled untagged product resulting from the induced hydrolysis from the tagged product and the unchanged labeled and tagged glycosaminoglycan substrate; (e) glycosaminoglycans in the human biological sample; measuring the amount of labeled untagged product that is proportional to the noglycan endoglycosidase concentration. (35) The method of claim (34), wherein the separating step comprises the use of antibodies raised against the tagged molecules or the use of binding proteins that bind to the tagged molecules. (36) The method according to claim (35), wherein the antibody or binding protein is bound to a solid support. (37) A buffered aqueous solution of a human biological sample suspected of containing glycosaminoglycan endoglycosidase is mixed with an antibody raised against glycosaminoglycan endoglycosidase, and the level of binding of said antibody to the sample is determined by An immunoassay method for detecting the presence of glycosaminoglycan endoglycosidase, the method comprising measuring as an indicator of the presence of saminoglycan endoglycosidase. (38) The method according to claim (37), wherein the antibody is monoclonal. (39) The method according to claim (37), wherein the immunoassay is a radioimmunoassay, an enzyme immunoassay, or a fluorescence immunoassay. (40) producing a liquid phase substrate that yields a labeled product when hydrolyzed by heparan sulfate endoglycosidase;
A method of assaying human heparan sulfate endoglycosidase using said substrate, comprising: (a) labeling heparan sulfate at one or more sites with a label that produces a detectable signal; (b) labeling the labeled heparan sulfate with a label that produces a detectable signal; (c) incubating the labeled and tagged heparan sulfate with a human biological sample suspected of containing heparan sulfate endoglycosidase; (d) the labeled and tagged heparan sulfate endoglycosidase. separating a labeled untagged product resulting from heparan sulfate endoglycosidase-induced hydrolysis of heparan sulfate; (e) an amount of labeled untagged product proportional to heparan sulfate endoglycosidase concentration in the human biological sample; A method including steps for measuring. (41) The separation is achieved by an antibody raised against the molecule used as a tag or by a binding protein that binds to the molecule used as a tag.
0) The method described in section 0). (42) The method according to claim (41), wherein the antibody or binding protein is bound to a solid support. (43) A buffered aqueous solution of a human biological sample suspected of containing heparan sulfate endoglycosidase is mixed with an antibody raised against heparan sulfate endoglycosidase, and the level of said antibody bound to the sample is determined to be An immunoassay method for detecting the presence of heparan sulfate endoglycosidase, the method comprising measuring as an indicator of the presence of heparan sulfate endoglycosidase. (44) The method according to claim (43), wherein the antibody is a monoclonal antibody. (45) The method according to claim (43), wherein the immunoassay is a radioimmunoassay, an enzyme immunoassay, or a fluorescence immunoassay. (46) A kit useful for detecting glycosaminoglycan endoglycosidase in a sample, the kit comprising: (a) a carrier compartmented to receive one or more container members in a sealed manner; a carrier; (b) a first container member containing an antibody specific for glycosaminoglycan endoglycosidase; and (c) a second container member containing a detectably labeled antibody specific for glycosaminoglycan endoglycosidase. Kit including parts. (47) A kit useful for the detection of glycosaminoglycan endoglycosidase in a sample, which comprises: (a) a compartmented carrier for receiving one or more container members therein in a sealed manner; (b) a first container member containing an antibody specific for glycosaminoglycan endoglycosidase; and (c) a second container member containing a detectably labeled glycosaminoglycan endoglycosidase. (48) Claim (46) or (48), wherein the antibody in the first container member is immobilized on the container member.
47) The kit described in item 47). (49) The kit according to claim (46), wherein the antibody in the second container member is labeled with a radioactive label, an enzyme label, a fluorescent label, or a chromophore. (50) Claim No. (46), wherein the antibody in the first container member or the second container member is a monoclonal antibody.
Kit as described in section. (51) The kit according to claim (46) or (47), comprising a number of container members having various amounts of glycosaminoglycan endoglycosidase antigen. (52) the first container member or the second container member is a tube;
The kit according to claim (46) or (47). (53) A kit useful for detecting heparan sulfate endoglycosidase in a sample, comprising: (a) a carrier compartmented to receive one or more container members in a sealed manner; (c) a second container member comprising a detectably labeled antibody specific for heparan sulfate endoglycosidase. (54) A kit useful for detecting heparan sulfate endoglycosidase in a sample, comprising: (a) a carrier compartmented to receive one or more container members in a sealed manner; A kit comprising: a) a first container member comprising an antibody specific for heparan sulfate endoglycosidase; and (c) a second container member comprising a detectably labeled heparan sulfate endoglycosidase antigen. (55) Claim (53) or (54), wherein the antibody in the first container member is immobilized on the container member.
) Kit described in section. (56) The kit according to claim (53), wherein the antibody in the second container member is labeled with a radioactive label, an enzyme label, a fluorescent label, or a chromophore. (57) The kit according to claim (53), wherein the antibody in the second container member is a monoclonal antibody. (58) The kit according to claim (53) or (54), comprising a number of container members having various amounts of heparan sulfate endoglycosidase antigen. (59) Claim No. (53) wherein the container member is a pipe.
) or (54). (60) When a glycosaminoglycan substrate is hydrolyzed by a glycosaminoglycan endoglycosidase, it yields a soluble product containing a label and a soluble product containing a tagged molecule, and the soluble products are a tagged molecule and a tagged molecule. 1. A method for producing a soluble glycosaminoglycan substrate comprising labeled and tagged molecules, comprising a non-hydrolyzed glycosaminoglycan substrate that can be extracted from solution upon exposure to a solid phase protein having binding affinity for (a) A label that produces a detectable signal at one or more sites on a glycosaminoglycan substrate for a glycosaminoglycan endoglycosidase and that does not substantially interfere with glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate. (b) labeling an unlabeled site of the labeled glycosaminoglycan substrate with a tagged molecule that has affinity for a protein capable of binding to the solid matrix; attaching a tagging molecule that does not substantially interfere with glycan endoglycosidase-induced hydrolysis. (61) A method for assaying glycosaminoglycan endoglycosidase, comprising: (a) applying a glycosaminoglycan substrate for glycosaminoglycan endoglycosidase at the above site;
(b) labeling the glycosaminoglycan substrate with a label that produces a detectable signal and does not substantially interfere with glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate; a tagged molecule that has an affinity for a protein that is capable of binding to a solid matrix and that does not substantially interfere with glycosaminoglycan endoglycosidase-induced hydrolysis of the labeled tagged glycosaminoglycan. (c) incubating the labeled and tagged glycosaminoglycan substrate with an aqueous buffer containing a human biological sample suspected of containing a glycosaminoglycan; (d) a glycosaminoglycan substrate; separating a glycosaminoglycan substrate and tagged product resulting from noglycan endoglycosidase-induced hydrolysis from an untagged labeled product resulting from the hydrolysis, the glycosaminoglycan-binding tagged molecule comprising: a separation step comprising: binding to a protein that has an affinity for the tagged molecule. (62) When a heparan sulfate derivative is hydrolyzed by heparanase, it yields a soluble product containing the label and a soluble product containing the tagged molecule, and when the soluble product is exposed to a solid-phase protein that has binding affinity for the tagged molecule, it goes into solution. 1. A method of producing a soluble heparan sulfate derivative comprising a label and a tagged molecule, including a tagged molecule extractable from a heparanase, comprising: (a) binding a heparan sulfate substrate to heparanase at one or more sites to produce a detectable signal; labeling the heparan sulfate substrate with a label that does not substantially interfere with heparanase-induced hydrolysis of the heparan sulfate substrate; (b) binding a tagged molecule to an unlabeled site of the labeled heparan sulfate substrate to the solid matrix; binding a tagged molecule that has binding affinity for a protein that does not substantially interfere with heparanase-induced hydrolysis of a heparan sulfate substrate. (63) A method for detecting heparanase, comprising: (a)
labeling the heparan sulfate substrate for heparanase at one or more sites with a label that has a detectable signal and does not substantially interfere with heparanase-induced hydrolysis of the heparan sulfate substrate; (b) labeled heparan sulfate; attaching to an unlabeled site of the substrate a tagged molecule that has binding affinity for a protein capable of binding to the solid matrix and does not substantially interfere with heparanase-induced hydrolysis of the heparan sulfate substrate; (c) incubating the labeled and tagged heparan sulfate substrate with a buffered aqueous solution containing a human biological sample suspected of containing heparanase; (d) the heparan sulfate substrate and tagged product resulting from heparanase-induced hydrolysis; separating the tagged molecule from the untagged labeled product resulting from hydrolysis, the separation step comprising: binding the tagged molecule to a protein having binding affinity for the tagged molecule. (64) The method according to claim (61) or (63), wherein the protein having binding affinity for the bound tagged molecule is bound to a solid matrix. (65) A kit useful for detecting glycosaminoglycan endoglycosidase in a sample, the kit comprising: (a) a carrier compartmented to receive one or more container members in a sealed manner; (b) a first container member comprising a substrate for glycosaminoglycan endoglycosidase and supporting a tagged molecule and a detectable label; and (c) a substrate having a specific binding affinity for the tagged molecule of the substrate. a second container member containing a protein. (66) The kit according to claim (65), wherein the protein is a monoclonal antibody. (67) Claim 6, wherein the monoclonal antibody in the second container member is immobilized on the container member.
The kit described in section 6). (68) The kit according to claim (65), wherein the detectable label is a radioactive label, an enzyme label, a fluorescent label, or a chromophore. (69) Claim No. 3, wherein the protein is an antibody (
65) The kit described in item 65). (70) The kit according to claim (65), comprising a plurality of container members having various amounts of glycosaminoglycan endoglycosidase antigen. (71) The kit of claim (65), comprising a number of container members having varying amounts of detectable labels. (72) Claim No. (65) wherein the container member is a pipe.
) Kit described in section.