JPH05509403A - Immunochemical localization of heparanase in mouse and human melanomas - 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] Determination of heparanase in mouse and human melanomas by immunochemical methods The U.S. government provides location determination services such as Rol-CA41524, and P30-CAI 6672, etc., which received the National Institutes of Health (NIH) ground. and reserves the rights to this invention.

Cross-reference of related applications This application is a partial continuation of application number 839.890 (7) filed on March 1986. No. 012,860, filed February 20, 1987. Co-pending U.S. Patent Application No. 377.0, filed July 7, 1989, which is a division. No. 15 is a pending application.

The purpose of the present invention is to investigate glycosaminosynthesis in various human and mouse tumors. The location of hevaranase, hevaranase, was determined by immunochemical methods.

The present invention also provides a method for quantifying endoglycosidase enzyme activity and a method for use in the assay. It also relates to labeled substrates.

The quantitative method of the present invention is also considered useful for detecting the malignancy of cancer.

A group of biological substances called proteoglycans are found in the extracellular matrix of connective tissue. It forms the basic substance in the water. These proteoglycans are high molecular weight polyaniline It is a chemical compound that has different types of molecules covalently bonded to the polypeptide backbone. Contains a heterosaccharide side chain. More than 95% of these proteoglycans It may contain carbohydrates. The polysaccharide groups of proteoglycans were previously They were all called mucopolysaccharides, but they all contain glucosamine or Because they contain lactosamine, they are now called glycosaminoglycans. It's been revealed.

Various enzymes are thought to be involved in the normal metabolic degradation of proteoglycans. available. The first step in proteoglycan degradation involves separating and cleaving the protein components. Includes proteolysis. As a result of this protein degradation, glycosaminoglycans are produced. be born. Next, glycosaminoglycan is glycosaminoglycan endoglyco It is broken down into smaller glycosaminoglycan fragments by the enzymatic action of sidases. It is decomposed by Glycosamino from glycosaminoglycans or their fragments The enzymatic action of glycan endoglycosidase depletes glycosaminoglycans. Monosaccharide is generated from the reducing end.

Endoglycosidases may be associated with tumor invasiveness and metastatic properties Therefore, interest in these enzymes has increased in recent years. N1col son (1982, Biochem, Biophys, Acta, V695 ．． PP I 13-176) has been reported to be associated with malignant tumors. We have written a review on various oligosaccharide-degrading enzymes R (PP141-142). I'm there. N1colson (1982, J,) Iistochem, & C ytochem, V30. PP 214-220) is a tumor of the endothelial cell basal lamina. Mechanisms proposed for tumor cell invasion and proteins and glycosaminoglycans Describes the degradation products from. Kramer et al.

(1982, J, Biol, chem, V257. PP2678-2686) is a fragment specifically enriched in glycosaminoglycans and free heparan sulfate. reported a tumor-derived glycosidase that can cleave .

Irimura et al. (1983, Analyt, Biochem, V30.

PP461-468) is a high-performance gel permeation chromatograph of glycosaminoglycans. It describes about Heparan sulfate degrading activity of melanoma cells was measured by this chromatographic method. Nakajima et al. (198 3, 5science, V220. PP611-613) in melanoma cell line This article describes the correlation between translocation activity and heparan sulfate degrading activity. Takamin Polyacrylamide gel electrophoresis, staining and density analysis of molecular weight heparan sulfate disappearance. It was tracked by tometry (densitometric method).

Vlodavsky et al, (1983, Cancer Res, V 43.

PP2704-2711) is a cluster of endothelial cells produced by two T-lymphoma cell lines. describes the degradation of 3″S-labeled proteoglycans from cells. Is the active system much more active in releasing cleaved 25S than the system with low metastatic ability? It was.

Irimura et at, (1983, Proc, Am, Soc, Canc er　Res.

V24. P37. abstract l 44) is a high performance liquid chromatograph. Describes heparan sulfate degrading enzyme activity in melanoma cells using the rough method. . Nakajima et at.

(1984, J. Biol, chem, V259. PP2283-2290) The properties of metastatic melanoma hebalanase are described. High performance gel permeability Functional substrates and products are described using romatograph methods and chemical analysis.

The background described here is that endoglycosidases play a crucial role in the formation of cancer metastases. This may be useful, accurate and I am interested in quantifying with good reproducibility.

The most important aspect of cancer cells is their ability to invade host tissues and metastasize to distant specific organs. It is one of the characteristics of Metastasis formation is a complex interaction between cancer cells and normal host tissues and cells. occurs through the unique interaction of chains. These processes are divided into several separate selections. Involves selective steps: invasion of surrounding tissues, penetration of vasolymphatic transport in the lymph or blood, or dissemination into the serous sinuses, arrest and distant location. The tumor invades the site and survives and proliferates to create secondary lesions.

The basement membrane contains collagen-like and non-collagen-like proteins and underlying parenchymal cells. extracellular matrix consisting of proteoglycans that separates from connective tissue It is a continuous sheet of They develop unique permeability and play a role in maintaining tissue structure. is fulfilled. Metastatic tumor cells rely on epithelial and endothelial basement membranes for invasion and metastasis. This penetration and destruction of the basement membrane by the invasive tumor cells can be observed using electron microscopy. Observed using a microscope. The basement membrane consists of type ■ collagen, laminin, and hepara sulfate. (H3), a polymeric unit containing proteoglycans and fibronectin It is a rigid structure consisting of a set of probably requires one or more tumor cell-associated enzymes to be active there. .

The unique physical and chemical properties of polyanionic and polymeric (Kanwar et at., 1980 J, Ce11) ．． Biol, V86. PP688-693), heparan sulfate (H3) is basal It is an important component of membranes. H3 is fibronectin, laminin, and type II cola These molecules are then transported into the basal lamina using antibodies made against each component. are observed together. H3 proteins collagen-like and non-collagen-like proteins. by promoting their interaction while protecting against the attack of degradative enzymes. It contributes to the assembly of the plate matrix. Therefore, the HS proteoglycan barrier destruction of the basement membrane is thought to be important for invasion of the basement membrane by tumor cells.

Interactions between malignant cells and vascular endothelium result in the formation of an extracellular matrix similar to basement membrane. This study has been carried out using cultured vascular endothelial cells called Zori. Using this model, Metastatic 816 melanoma cells contain matrix glycoproteins such as fibronectin. Degrading matrix sulfated glycosaminoglycans such as heparan sulfate and heparan sulfate was discovered. Heparan sulfate is fragmented into fragments approximately one-third of its original size. Metastatic tumor cells are characteristically endocytosed with heparan sulfate because they are liberated in solution. It has been thought to contain glycosidase.

A transfer characterized by enzymatic degradation of the subendothelial extracellular matrix. Invasive potential of five B16 melanoma sublines that differ in terms of engraftment and invasion What was not found in relation to metastatic potential is that B16 sublime cells, which have high invasive potential and metastatic potential, Purine degrades sulfated glycosaminoglycans more rapidly than Purine, which has a lower metastatic potential. (Nakajima et al., (1983), 5ci ence V 220.

P611) and intact 816 cells (or their absence) with high lung colonization potential. Cell homogenate) also purified sulfuric acid compared to 816 cells, which have low lung colony forming ability. This means that heparan is degraded more quickly (ibid.). Seed by 816 cells HS and other purified glycosaminoglycans from various sources (heparin, chondroi, etc.) Chin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and The ability to decompose hyaluronic acid (hyaluronic acid) was investigated.

High-performance gel permeation chromatography was used to analyze glycosaminoglycan degradation products. An analytical method using the fu method was developed (rrimura et al., (19 83) Anal, Biachern.

Vl　30. PI 61; Nakajimaet al, (1984) J, Biol, Chem, V259. P 2283). (”S　) Metabolized by sulfuric acid HS labeled with EHS sarcoma and PYS-2 carcinoma cells, and in the calf aorta. Purified from the basement membrane that produces corneal epithelial (EAE) and corneal endothelial (BCE) cells. (ibid.). 83 molecules purified from bovine lung and other glycosaminoglycans The reducing ends of these molecules were labeled with tritium using N a B (”H) a. Ta. Glycosaminoglycans labeled in this way are 8 in the presence or absence of D-sugar acid 1,4-lactone, a clonidase inhibitor. 16 cell extracts and separated by high performance gel permeation chromatography. The decomposition products were analyzed. The above-mentioned H3s isolated from various sources are all characteristic molecules. It was broken down into fragments with a certain amount. In contrast, hyaluronic acid, Leutin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate and heparin were hardly degraded. Heparin and other glycosaminoglycans Unlike KAN, it inhibited H8 degradation. The decomposition of H3 into specific molecular weight fragments The time-dependent effect shows that melanoma henocyclase binds H3 at specific chain positions. It shows that it is disconnected (same as above). To determine the specific H3 cleavage point, The newly generated reducing end of the HS fragment was examined as follows: Na B C”H) Labeled with a; Hydrolysis to monosaccharide: and paper -Analysis of these satucharides by chromatographic methods. from HS fragment The 3H-reducing terminal monosaccharide is overwhelmingly (>90%) L-gulonic acid. Therefore, the H3-degrading enzyme involved here is endoglucuronidase (hebalanase). ze).

H3-degrading endoglucuronidase was detected in human dermal fibroblasts, rat hepatocytes, and human It has been found in a variety of tissues, including human placenta and human platelets. H in mammalian cells Other studies on S-resolving endoglucuronidase (heparan sulfate) - “heparitinase” which indicates a specific endoglycosidase reported.

However, heparitinase was originally produced by Flavobacterium heparj This is the name used to name the removal enzyme (EC4, 2, 2, 8) in nus. This enzyme produces H3 unsulfated and monosulfated 2-acetamido-2-deoxy-α - Cleave the D-glucosyl-D-hexuronic acid linkage. in mammalian cells HS-specific endoglycosidases except those found in skin fibroblasts Since it is an endoglucuronidase, it is a mammalian cell enzyme that can degrade HS. Doglucuronidase is in contradiction to the currently used term “heparan sulfate.” It should be called ``hebaranase'' so as not to shield it.

The enzymatic activity of glycosaminoglycan endoglycosidases can be determined in several other ways. Quantitated. For example, 01dberg et al. (1980, Bi ochem, VI 9. PP5755-5762) is heparin-like polysaccharide describes the quantification of platelet endoglycosidase, which degrades . This quantitative method measures the amount of decrease in 8H-heparan sulfate; It is a function of endoglycosidase activity.

About the quantitative method of endoglycosidase using a solid phase substrate [verius (1 971, Biochem, J, VI 24. PP677-683) and 0os ta et al. (1982, J. Biol.

Chem, V257. PPI 1249-11255). rve rius synthesizes various glycosaminoglycans with cyanogen bromine-activated Sepharose. 4B beads. In one case the endoglycosidase hyaluronida The enzymatic activity of this enzyme was determined by combining this enzyme with chondroitin sulfate bound to Sepharose 4B. was incubated with and quantified. Monitoring enzyme activity determines the production of soluble uronic acids. Follow-up was carried out using a colorimetric method. oosta et al, heparin and sulfate About heparitinase, an endoglycosidase in platelets that cleaves paran. Describes the quantitative method of

The system of 0osta et al. and its quantitative method are as follows. (1) Heparin is converted into N-succinimide 3-(4-hydroxyphenylpropion) Conjugate with an acid salt.

(2) Incubating conjugated heparin with Na''I and chloramine-T and mark it.

(3) ”’　I Heparin and Sepharose 4B cyanogen bromide activity (4) Conjugate endoglycosidase to Sepharose 4B beads. Beads were incubated with conjugated 12sI-heparin and then solubilized. Measure radioactivity.

In these two methods, glycosaminoglycans are prepared whose free amine groups are amino- Cross-linking is achieved by reacting activated cyanogen bromide with activated agarose. make.

Glycosaminoglycans such as heparin and heparan sulfate contain some free glycans. Since it has a cosamine amino group, when this type of crosslinking occurs, the substrate molecule and agarose This results in excessive co-growth between the base gel and the loss of sensitivity to endoglycosidases. Therefore, the decomposition has a nonlinear rate. Therefore, glycosaminoglycan endoglyco The most preferred solid phase substrates for sidases are those that are immobilized at one end of the molecule, such as the reducing end. It is a glycosaminoglycan cross-linked to a phase support.

Heparanase, or endo-β-D-glucuronidase, is associated with melanoma metastasis. has been associated. Polyclonal antibody against mouse N-terminal hebalanase 5DS-polyacrylamide, a cell lytic component of trout melanoma and human melanoma. A protein with Mr~97,000 was detected on dog gel electrophoresis. indirect immune cells In chemical studies, human A375-3M and mouse B16-BL6 melanoma cells stained with hevaranase antibody. Hebaranase antigen is the same as on the cell surface of non-permeable cells. Similarly, it was localized in the cytoplasm of exudative melanoma cells. B16-BL6 Melano Immunohistochemical staining of frozen sections from syngeneic mouse lungs containing tumor micrometastasis. We thereby demonstrated that heparanase was localized in metastatic melanoma cells. Is it a patient? A similar study using frozen sections of resected malignant melanoma revealed that invasive melanoma It was shown that heparanase is localized in cells. From various studies related to the present invention It is suggested that: (a) the N- The termini are antigenically related: (b) Heparanase antigens are metastatic human and mouse localized in the cell surface and cytoplasm of smelanoma cells; and (C) heparana -ase antigen is increased in invasive and metastatic mouse and human melanomas in vivo. It is growing. Polyclonal antibody preparations are used here, but monoclonal monoclonal antibodies or mixtures of monoclonal antibodies with the same specificity are also available using known methods. could be made and used in the same way if needed.

Soluble labeled products upon hydrolysis by glycosaminoglycan endoglycosidases A solid phase substrate that yields a solid phase substrate and a method of producing this substrate are included within the present invention. solid The phase substrate contains a labeled N-acetyl group and is reductively aminated at the reducing end to form an amine- Contains glycosaminoglycans that form the terminus. The substrate has its amine-terminus conjugated to an amino-activated solid phase matrix.

The method for producing a solid phase substrate consists of the following steps: N-desulfation or N-acetylation of glycans; at least partially Labeling glycosaminoglycans by labeling acetylated or desulfated glycosaminoglycans Making Noglycan: Labeled glycosaminoglycans are converted into acyl anhydrides or halogenated acyl complete N-acylation with sil; the reducing end of the labeled glycosaminoglycan above is Reductive amination creates labeled amine-terminated glycosaminoglycans. And the end The labeled amine-terminal glycosaminoglycan is transferred to the amine-activated solid phase through the terminal amine. Conjugate to a support to create a solid phase matrix substrate.

Partial N-desulfation or N-acetylation of substances that give a detectable signal Labeling is completed by substitution on the amino group of the glycosaminoglycan. this The substance is a radioisotope label, a fluorescent label or an enzyme label. Fluorescent labels are Radioisotopes are similar to natural glycosaminoglycans in that they are easy to quantify. It is excellent in what it does.

Figure 1. Synthesis of solid-phase heparanase substrates: chemical modification of H8 and radiolabeling and amino - Conjugation to activated agarose gel beads.

Figure 2.816 Unmodified and modified melanoma before and after treatment with hebalanase Chemical modification-H3 elution characteristics on high performance gel permeation chromatography. H3, sulfuric acid Paran; NDS, N-sulfated sulfated heparan NDS-HAc-H3, N-desulfated N-acetylated heparan sulfate. These tritium-labeled molecules at the reducing end (marked with ○) Glycans were converted to B16 melanosomes in the presence of sugar acid 1,4-lactone (SAL) (marked with a *). Fragments of these glycans obtained by incubation with mer cell extracts. , 0.2M sodium chloride Fractogel-TSK HW-55 (s ) on two consecutive 0.75X75-an columns at 55 °C with a flow rate of 0-7 min. A romatograph was performed.

The arrows in (a)-(e) are the elution positions of labeled glycans: (a) Shark cartilage (Mr C6S from bovine lung (Mr 60,000); (b) H3 from bovine lung (Mr 34,000 ); (C) Heparin from pig mucosal tissue (Mr 11.000): (d) Pig Monosialosylubiantennary complex type glycosylation from thyroid globulin Peptide (Mr 2190) + (e) N-acetyl-D-glucosamine (Mr 221).

Figure 3. Partially N-desulfated N(”C)acetyl immobilized on agarose Heparan sulfate (PNDS-N(”C)Ac-H3) to B18 melanoma cells Dose-dependent degradation by paranase. PNDS-N immobilized on agarose (1 4C) Ac-H3 (4500 cpm) was added to various amounts of 816 cells in the presence of SAL. Extract for 6 hours (mouth), 12 hours (○ mark), and 24 hours (△ mark), or Various amounts of heat-inactivated (100°C for 15 minutes) B16 cell extract and 12-hour ink. It was incubated. Added cell extract (μ g protein) was plotted.

Figure 4. The magnitude of heparanase activity in the serum of controls and malignant melanoma patients is shown.

Figure 5. Hebalanase activity in the serum of rats injected with highly metastatic adenocarcinoma Indicates size.

Figure 6. Correlation between serum heparanase activity and primary metastatic tumor size in rats Indicates a relationship.

Figure 7. Showing the correlation between rat serum heparanase concentration and the number of metastases from malignant tumors. vinegar.

Figure 8. The location of substrate hydrolysis for melanoma hevaranase is shown.

Figure 91 Human A375-SM metabolically labeled with (''S)-methionine (a-d) and mask B16-BL6 (e-h) immunoprecipitated from melanoma cell lysates. Autoradiograph of 7.5% 5DS-polyacrylamide gel containing protein I. Cell degradation products were added to PBS (d.

h), biotinylated anti-hevaranase antibodies (b, c.

f, g), or biotinyl incubated with heparanase peptide. anti-heparanase antibody (a.

e). Then add them to strebtoavidine-agarose. (a-h) and the samples were run in the presence of β-mercaptoethanol. I let it out.

The molecular weight marker far to the right is expressed in kDa.

Figure 10. Immunology of heparanase in human A375-5M melanoma cells Photomicrograph of tissue chemical localization. Cultured cells were fixed (a-f) or permeabilized ( a, C-f). They were then treated with anti-heparanase antibodies (a, b).

e), anti-hebalanase antibodies preabsorbed with hebalanase peptides (d) or or PBS (c, f). After that, wash the cells and apply Perio. Oxidase-conjugated goat-anti-rabbit IgG (a, b, d, f) or PBS (c, e). Original magnification factor x 25; bar = 30μmoE fine Mirror photos have the same magnification.

Figure 11. Human skin melanoma (a, b, c) or mouse melanoma in the lungs Immunohistochemical localization of heparanase in metastases (d, e, f) 02% Frozen sections fixed with paraformaldehyde were stained with hematoxylin and eosin. a, d) or anti-heparanase antibodies or heparanase peptides (b) or or with anti-hebalanase antibody preabsorbed in PBS(e). Ta. The sections were then treated with peroxidase-conjugated goat anti-rabbit IgG (b, c, e, f). Original magnification x 80: bar = 20 μm 0 microscope Microphotographs have the same magnification.

For example, glycosaminoglycan agents such as heparan sulfate endoglycosidase We will describe an immunoassay method using an antibody against glycosidase; It measures enzymes. In this application, glycosaminoglycan endoglycoside enzyme activity, most of which is derived from heparan sulfate endoglycosidase called hevaranase. Regarding the activity of enzyme, we will also discuss a new method for quantifying it. This new definition The quantitative method uses glycosaminoglycan endoglycosidase to hydrolyze soluble It is described using a solid phase substrate that produces a labeled product. This new quantitative method is based on this same phase fermentation. A new application of the quantitative method to liquid phase conditions is also discussed.

Among many cellular functions, the extracellular matrix plays a role in tumor cells or as a process of invasion and metastasis. It is an organizational barrier that must be penetrated by the process. Extracellular matrix is basement membrane It contains connective tissue stroma, collagen, proteoglycans, laminin, and fibers. Consists of fibronectin and other glycoproteins. Importance in tumor cell invasion and metastasis This matrix degradation, which is a critical step, involves proteins of tumor cell or host cell origin. Various degradative enzymes are involved, including rotease and glycosidase (Mull ins et al, Biochi [Il.

Biophys, Acta, 695. 177-214 (1983): Na Kajima et al., J. Ce11. Biochem, 36, 157-167 (1988); N1colson, Biochim, Bio phys, Acta, 695, 113-176 (1982); Liot ta, CancerRes.

46, 1-7 (1986); 5loane et al, cancer Met.

Rev., 3, 249-263 (1984). 1 extracellular matrix-min The degrading enzyme hebalanase was first found in mouse and human melanoma cells ( Kramer et al.

J. Biol. Chem., 257. 2678-2686 (1982); Nakajima et al., 5science, 220. 61 1-6 13 (1983), the enzyme was purified as a glycoprotein of Mr~97,000. Specialized (Nakajima et al, J.

Ce11. Biochem, 36. l 57-168 (1988), Hepa The magnitude of lanase activity was determined from mouse metastatic melanoma cells (Nakajjma et al. al,, 5science, 220. 611-613 (1983) and Hitome Ranoma cells (Nakajima et al, cancer Lett, 31. 277-283 (1986a), which is directly related to the lung-colonization ability of Such activity was found in the serum of melanoma patients with metastatic disease (Naka et al. jima et al, J, cell, Biochem, 36, 157- 167 (1988). Heparaners such as heparin and its chemically modified derivatives 816 mouse melanoma in experimental metastasis quantification. Inhibits lung metastasis (Irimura et al., Biochemistry , 25, 5322-5328 (1986); Villanuevae tal,, I　988). Therefore, tumor cell heparanase is involved in melanoma invasion. It seems to play an important role in attack and metastasis.

The role of degradative enzymes in the process of tumor invasion and metastasis has been studied (Mullins et al, Biochim.

Biophys, Acta, 695. 177-214 (1983); Li otta et al, Cancer Res, 46. l-7 (1986 ); N1co1son, Biochim, Biophys, Acta, 695 , 113-176 (1982), the location and origin of these enzymes are currently unknown. Until then, little attention had been paid to it. What information is currently available about this? is mostly cathepsin B (Sloane et al., cancer Met ,Rev,,3. 249-263 (1984) and solid collagenase (Lio tta et al.

Nature (Lond) 284. 67-68 (1980), etc., Protea (Moscatelli et al., Biochim.

Biophys, Acta, 948. Based on 67-85 (1988) There is. These enzymes were analyzed using biochemical analysis of enzyme activity in subcellular fractions. The relationship between this and cell surface membranes and various cellular components has been clarified. Cathepsin B activity is detected in the lysosomal and plasma membrane fractions of B16 mouse melanoma cells. (Kerem et al, CancerRes, 48.1461- 1421 (1988); Rozhinetal, cancerRes, 4 7. 6620-6628 (1987), and the enzyme can also be transduced by viruses. It was localized on the cell surface of formed fibroblasts and other cells (Sylven et al, Virchows Arch, B Ce1l Pathol, , l　7゜97-112　(1974). Hebalanase activity is measured on the melanoma cell surface. It has been found in cell homogenates and shed vesicles of melanoma cells (N akajima et al.

5science, 220, 611-613 (1983), Immunology related to degradative enzymes. Epidemiological studies have provided more detailed information about the localization of these enzymes; This information can be used to determine the origin of enzymes in tumors, including normal host cells, and to identify fibroblasts. It is particularly useful for defining structures such as cells and lymphocytes. Cathepsin B is a rabbit It was localized on some normal cell components in the invasive front of gigantoma (Graf etal, , Lab, Invest, , 45. 587-596 (1981 ).

Anti-heparanase antibodies directed against N-terminal heparanase peptides according to the invention was prepared and its properties were described. Heparanase was tested in humans and humans using indirect immunochemical methods. It was confirmed that the protein was localized in the cytoplasm and on the cell surface of mouse melanoma cells.

Antibodies have been tested in mouse experimental metastasis models and in human melanoma from patients with metastatic disease. The metastatic melanoma nodule within was strongly stained.

This solid phase substrate contains a glycosaminoglycan with a radiolabeled N-acyl group. I'm here. These labeled N-acyl groups are preferably 3H-labeled or 14C-labeled acetyl groups. However, other labeled acyl groups such as formyl or propionyl groups may also be used. Book The solid phase substrate of the invention contains hyaluronic acid and chondroitin as glycosaminoglycans. 4-sulfuric acid, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, hepasulfate Contains lan, heparin or a combination thereof. certain glycosaminogs By using lycan, the substrate for the specific glycosaminoglycan used is It will be possible to quantify the enzymatic activity of active endoglycosidase.

The amino-active solid polymer to which the amine-terminated glycosaminoglycan will be attached Trix is based on both the basic properties of Matri-Sox and the amine-active chemical position. Both may have many receptive types.

Preferred solid matrices are agarose-based, with the most preferred being What is important is Sepharose or Sepharose derivatives in bead form (Pharmacia ).

Cellulose or polyacrylamide and other solid matrices are If it has an amine-activated substitution function for the role, it could be used.

Sepharose beads are activated with cyanogen bromide to release heparin and other glycosaminoglycans. Conjugate to molecules with amines such as lycans or glycosaminoglycan derivatives It is well known that this will happen. Conjugation mediated by cyanogen bromide is preferred for the practice of the present invention. It is a useful conjugate for cyanogen bromide activated agarose or other Glycosaminoglycans with multiple amine functionalities even in active amine-active solid matrices may be conjugated with one or more amine groups of can and glycosaminoglycan derivatives. cormorant. This multiple conjugation to labeled glycosaminoglycans This can make glycosidase quantification insensitive and/or inaccurate. and This is because a military-glycosidase-mediated proteolytic event produces one product, even This is because it may not produce products that are not bound to the solid matrix. . Therefore, for carrying out the present invention, only one labeled glycosaminoglycan derivative is required. It is important to bond to a solid matrix in which primary amino groups are present.

A variety of amine-activated substituents are known to those skilled in the art; N-hydroxylated succinide esters are the preferred amine- It is a functional group and is commercially available or can be easily synthesized. Such N-hydroxylation Succinide esters are conjugated with primary amine groups at pH between about 6 and 9. agaro The base is activated by periodic acid oxidation to have an aldehyde functionality. child The aldehyde agarose reacts with the labeled amine-terminated glycosaminoglycan and its The chain is stabilized by reduction with sodium cyanoborohydride (Perikh et al,, Methods in Enzym, Vol XXXW, P 8 1 Acad, Press (1974), Labeled glycosaminoglycerides with amines Other methods that may be used to bind lycans to solid matrices Commonly used procedures include dicarbodiimide and carboxy- Using a solid matrix containing amine-containing labeled glycosaminoglycans directly with solid matrices with moacetyl, diazonium or epoxy functional groups. make contact with it.

Glycosaminoglycans generally have their amine functional groups sulfated or acetylated. has been done. For example, after partial N-hypothesis sulfation or N-hypothesis acetylation, the result The primary amino group formed as is used for labeling.

Deacetylation may lead to glucosaminyl linked hybrotysls. This is done by hydrazinolysis under conditions that avoid excessive alkalinity. Detachment Sulfuric acid is used to make pyrimidinium salts of glycosaminoglycans, and then dimethylsulfo It can be done by solvolysis in SIDS. The amine group is labeled with fluorescein iso Fluorescent compounds such as thiocyanates, alkaline fusphatase (and bifunctional with enzymes or radiolabeled acyl anhydrides or acyl halides, such as It is done by reacting. The label is then covalently bound to at least some of the free amino groups. match.

The remaining free amino groups of the labeled glycosaminoglycans are then treated with e.g. acetic anhydride. Acylated during processing. The acylated labeled glycosaminoglycan is then The reducing end is aminated. This amination creates a Schiff base, which is then reduced by incubation with an amine salt to create a terminal amine. .

Amino group labeling is used to label measurable compounds or active proteins with at least one amino group. It is carried out in conjunction with shoes. Visible light is very effective for measurable compounds. one of the many compounds known to be absorbed, or more preferably Examples include fluorescein, which is excited by irradiation with a specific wavelength and fluoresces. There are substances that emit Enzymes (alkaline phosphatase mentioned above) as active proteins Glycosaminoglycans that are partially N-desulfated or N-acetylated Labeling by binding to cans is also one possible method. However, enzymes or light Compounds that absorb or can be measured because of their fluorescent structure contain sterically large substituents. there is a possibility. Such sterically large substituents, if too many are substituted, Glycosaminoglycan derivatives against glycosaminoglycan endoglycosidase This would make it an insufficient substrate for use. In preliminary experiments, partially N-desulfated heparan sulfate is 1=1 with fluorescein isothiocyanate. conjugated at a ratio of This fluorescein-labeled derivative is a melanoma hebalanase. It turned out to be a good substrate. The ratio of fluorescein to Is is lo:1. It is expected that it will serve as a substrate for heparanase.

Only one or a few of these large substitutions result in good labeling without interfering with substrate activity. Can be a substrate. Another problem that can arise with enzyme labels is the general Enzymes have free amino groups that bind to amine-active solid matrices. It may be possible to match. One preference for glycosaminoglycans The new label is a radioisotope label that is structurally identical to the natural N-substitution. ! sS -N-substituted sulfuric acid can be used, but it is difficult to make "C- or 3H" -acetyl N-substituted product is preferred. The substrates and methods described below are primarily radioactive Although related to topical labeling, it can basically be applied to other labels, especially fluorescent labels. This is Shiuruchino.

N-radioisotope-labeled glycosaminoglycans in a solid matrix Glycosaminoglycan endoglycosidase when attached by a terminal linkage A solid phase substrate is created for. As mentioned elsewhere in this specification, this solid phase group The quality of soluble glycosaminoglycans as a function of endoglycosidase enzyme activity. Generates radioisotope labeled substances. An alternative way to measure this same activity is to measure enzyme activity. By observing the disappearance of solid matrix-bound radioisotope labels as a function of Probably. This type of measurement has the disadvantage of being a negative measurement, and also has the disadvantage of being a negative measurement. The incubation supernatant should be carefully removed from the residual solid matrix substrate. There is also the disadvantage of being

In a broad sense, the solid phase substrate of the present invention is suitable for glycosaminoglycan endoglycosidases. hydrolysis to yield a soluble product labeled with a detectable signal. It is something like that. This solid phase substrate is suitable for glycosaminoglycan endoglycosidase. glycosaminoglycans with a label that does not interfere with the hydrolysis of labeled glycosaminoglycans by Contains glycans. Labeled glycosaminoglycans are preferably It is attached to the solid matrix by a covalent bond at its reducing end. detectable Gunals may be radioactive, light-absorbing, fluorescent or enzymatically active. The solid matrix is the parent Aqueous or preferably cellulose, dextran, polyacrylate or the like Contains derivatives or combinations. Is the substrate of the present invention a label to be detected? It will be soluble if present together with the identification molecule.

The identification molecule is a “handle” for removing the bound glycosaminoglycan portion. used as.

Labeling of at least partially desulfated or deacylated glycosaminoglycans is , acetyl halides, especially chlorides or alkyl bromides, but are most preferred. Preferably, this is carried out by treatment with 2H-acetic anhydride or 14C acetic anhydride. centre In addition to other acyl functional groups, such as formyl or propionyl, other conjugation methods are available. It may be used in this labeling method.

The substrate of the present invention is a liquid phase substrate that separates the unreacted substrate and cleavage product generated after the enzymatic reaction. There is also. In this scheme, glycosaminoglycans, such as heparan sulfate, KAN can be labeled to other molecules at its terminus, preferably the reducing end. Glico Saminoglycans are further processed by other molecules such as ItsI, fluorescein, and enzymes. should be labeled at an additional position, which is useful for detection of cleavage products in quantitation. Sometimes used for. The advantages of using liquid substrates of the type described here and is a highly sensitive assay for the action of glycosaminoglycan endoglycosidase. The amount is what you can do. This high sensitivity is at least partially due to solubility in solution. This will expand the use of enzymes.

The molecular label on one end of the glycosaminoglycan is fluorescein or biotin. It can be a small molecule such as a peptide or a large molecule such as a peptide or protein. It can be a child. The attachment of this molecule to the end of the glycosaminoglycan substrate is thereby Glycosaminoglycan labeled with glycosaminoglycan endoglycosidase It must not significantly inhibit the hydrolysis of water. Molecular beacons are labeled Furthermore, for glycosaminoglycan chains, glycosaminoglycan endog For the residues of glycosaminoglycan chains left after cleavage by lucosidase, It should have the ability to act as a potential handle.

As a handle, the molecule is a protein with an affinity for the bound label molecule. It could serve as an attachment point for white matter molecules. Such proteins- Molecular interactions allow labeled glycosaminoglycan attachment to become endoglycosylated. Glycosaminoglycan substrate hydrolysis liberated by sidase-induced hydrolysis can be easily separated from the labeled but unattached portions.

The molecular attachment must be either a) a carrier such as a protein; When administered to animals immunologically, antibodies that specifically bind can be produced. habten-like molecules = b) global immune substances such as proteins or peptides or or C) for example into avidin or a protein. A substance that has strong binding affinity for existing protein-like molecules.

After incubating the sample with endoglycosidase activity, the uncleaved product for example, by incubating with a solid-phase antibody that has an affinity for the attached material. will be separated from the cleavage products by Attaches to the ends of glycosaminoglycans. Proteins other than antibodies can also bind to uncleaved glycosaminoglycans. It can be used to separate. When using solid-phase antibodies or solid-phase binding proteins, If the solid phase is something that is easily conjugated or absorbed by the antibody or binding protein, It appears that the cleavage product is separated from the uncleaved substrate. It must have an impact on. Examples of commonly used solid phases are agarose: Polymers such as sepharose and polystyrene: glass; cellulose and glass peas and magnetized beads. large or small particles in solid phase form; tubes or microtiter plates, or their equivalents that easily fit into the detection system. There could be others.

Immunoprecipitation was used to separate uncleaved and cleaved glycosaminoglycan products. It can also be carried out using a solid phase-bound antibody (M organ et al, (1962) Prac, Soc, of Exp, B iol,Med,.

Vl　10. PP29-35), the precipitating antibody is attached to the end of the glycosaminoglycan chain. It should react with molecules that land on it.

To facilitate attachment at one terminal position of the solid matrix, labeled glycosa Further modification of minoglycan is required. One of these modifications is labeled glycosaminoglycerides. Substitution of an amino group at one end of the lycan. Preferred for making this replacement The method involves converting labeled glycosaminoglycans into ammonium salts and cyanoborohydra. and incubate at alkaline pH. Then, first, ammonia A ship base is formed between the a and the aldehydic carbonyl group of the terminal hexose.

This Schiff base is reduced by sodium cyanoborohydride to form a primary atom. Become Min.

The synthetic steps for producing the solid-phase substrates of the invention generally include, for example, hydrazinolysis. N-hybridization by acylation or solvolysis in dimethyl sulfoxide Desulfation; N-acyl anhydride or halogen-based N-acyl anhydride for radiolabeling. Reductive amination step: and the newly introduced terminal amine Including conjugation to a solid matrix through. Amine-antibody on solid matrix The preferred final step to ensure that no reactive functional groups remain is to Matrix-labeled amine-terminated group conjugated with umcyanoborohydride By incubating compounds with free amino groups with lycosaminoglycans, be. The latter compounds include, for example, ethanolamine or glycine ethyl There's Esther.

The substrates and methods of the invention are useful for determining glycosaminoglycan endoglycosidase enzyme activity. Offers many advantages in terms of quantity. For example, the substrate of the present invention may be A linear pattern of enzymatic produces a product.

Until now, proteoglycans containing glycosaminoglycans as well as binding protein components have been proposed. Can is bound to a solid matrix of cyanogen bromide-activated agarose. Ta.

In that case, proteoglycans bind to agarose primarily through their proteinaceous components. tried to match. Thus, proteolytic activity is linked to glycosaminoglycan endoglycosylation. Similar to sidase activity will liberate soluble products.

The specificity of the method for quantifying the enzymatic activity of endoglycosidase is greater than that shown in the present invention. It gets lower.

Heparanase obtained from human melanoma cell line (heparan sulfate endoglycosylation) Dase) can partially degrade N-desulfated, N-acetylated heparin to I understand. This same enzyme preparation converts N-desulfated heparan sulfate into N-desulfated N- Cuts into characteristic fragments as efficiently as acetylated heparan sulfate I found out that it does.

There are many types of glycosaminoglycan endoglycosidases, but hepatosulfate Heparan sulfate endoglycosidase, an endoglycosidase that uses orchid. Thus, heparanase was selected as a typical example showing a preferred embodiment of the present invention.

In addition, a preferred method for conjugating labeled amine-terminated heparan sulfate to create a solid phase substrate. N-hydroxylated succinide agarose derivative was selected as a new solid matrix. . Heparanase activity is shown to be specific for many of the following soluble A radiolabeled product was produced.

Melanoma hevaranase is an endo-β-glue that specifically cleaves HS at an intrachain position. It is clonidase. Figure 8 shows the specificity of melanoma hebalanase. It is. In this way, heparanase quantification can be performed using the reaction product, which also depends on its size. It is then necessary to separate it from the substrate. polyacrylamide gel electrophoresis Previously established methods such as high performance gel permeation chromatography and high performance gel permeation chromatography Although useful for separating samples, rapid and small-scale determination of large numbers of samples is Not suitable for quantitative assays. Bind covalently to perform rapid quantitative assays A well-treated substrate is required. The solid phase quantitative substrate developed in the present invention is Affi-Gel 1. Partial N-acetylation or N-desulfation conjugated with 5, N-(“Hor”C )-acetylated H3. In this substrate, the HS derivative is and is bound to agarose (Figure 1). This product is the most sensitive to be developed. It is one of the best endoglycosidase substrates. This substrate is currently available in mice and humans. It has been successfully used for ranoma hebanase quantification. The derivative of the same type is Rea It is also made using Cti-Ge 1 (NW-65F) (Pierce , Rockford, IL) a However, Af f 1-Ge 115 and Reac t1-Ge l (HW-65F) Both use fairly large particles. It retains a significant amount of high molecular weight material in the gel matrix for use. child may be a problem in some quantitative heparanase assays and Therefore, we use an Affi- A more desirable quantitative group using Gel 701 or 702 (Bio-Rad) Developed quality. The specific synthesis method is as follows. Sodium borohydride labeled H8 Reduction with dryide produces sugar alcohol at the reducing end. Excessive sugar alcohol - Converted to primary aldehyde by oxidation. This aldehyde group is then converted into a Schiff base. to beads coated with amino-derivatives such as Affi-Gel 701. combined and stabilized by reduction with sodium cyanoborohydride. death However, the method proposed further, which is similar to the method we developed before, is It is expected that it will be useful.

Radiolabeled HS with amino groups sulfated or acetylated is reduced under reducing conditions. The original end should be aminated with ammonia. Affi-Gel 702 is by N-hydroxysuccinimide or N,N-1carponyldiimidazole should be converted into amino-active beads by induction, followed by aminated radioactive H 3 should be coupled to amino-active Affi-Gel 702.

Bolton and f (Iodination of HS with Inter reagent may result in structural changes) Although disadvantageous due to the fact that the substrate is more radioactive with the use of 126I-labeled H3 It might be possible to do it.

On the other hand, for routine clinical research, fluorescein-free HS should be used. The quantification method would be improved by: fluorescein-labeled H3 could be It is suitable for rapid analysis of degraded fragments on an HPLC equipped with a detector.

Glycosaminoglycans such as heparan sulfate endoglycosidase (heparanase) The quantitative method for measuring the concentration of endoglycosidase also applies to endoglycosidase. immunoassay assay using polyclonal and/or monoclonal antibodies against - It may also be carried out using mats. Rather, Oldberg, et al. al (1980) Blochem.

V19. Like the platelet endoglycosidase described by PP5755-5762 Antibodies with relatively low cross-reactivity to other endoglycosidases can be used. . Antibodies can be used to directly measure endoglycosidase proteins using various immunoassay methods. used for. Endoglycosidase is described by Berson and Yalow ( 1968) Cl1n, Chem, Acta, V22, P51 Using immunoassay or 125I-labeled antigen or antibody, Miles, etc.

(1976) Anal, Biochem, V61. PP209-224? Determined by the immunoradiometric (IRMA) assay described. endoglycosidase may also be determined by enzyme-linked immunosorbent assay, or in this case by competitive-binding assay or is similar to IRMA, alkaline phosphatase, horseradish peroxidase , or WIsdorn (1976) Cl1n.

Chem,, V22. As reviewed by PPl243-1255 using an antibody or any other enzyme conjugated to an endoglycosidase. ``Sandwich'' quantitative method is used.

The subcellular localization of degradative enzymes involved in tumor invasion and metastasis has been investigated both biochemically and immunologically. studied epidemiologically. However, most of our current knowledge on this issue Based on research using proteolytic enzymes such as tepsin B and collagenase type II. Ru. 5 Loane and her joint research on the speed of sound (Rozhin et al, ca. ncer Res,, 47. 6620-6628 (1987) + 5lo ane et al, Proc, Nath-Acad, Sci.

83.2483-2487 (1986), Catalog of metastatic B16 melanoma cells. reported that pusin B-like activity was detected in plasma membrane-associated fractions. K eren etal, cancerRes, 48. 1416-1421 (1988) cell surface associated molecules of metastatic B16 melanoma and fibrosarcoma cells. that there is cathepsin B-like activity in a 2% butanol extract containing Plasma membrane-associated cathepsin B activity is associated with cancers such as melanoma, lateral strangulation and breast cancer. reported that it appears to be correlated with the metastatic potential of tumor cells, including cells. tumor Relationship between collagenase activity in the conditioned medium of cells and their invasive and metastatic potential There is a good correlation between them (Liotta et al., Nature (L ond, ) 284. 67-68 (1980): NakaJiIIIaet al,, cancerRes,, 47. 4869-4876 (1987) o Nakajima et al, Cancer Res, 4 9.　1　 6 98-1706 (1989) found that plasma membrane-associated 6 We have discovered a 4kDa ■ type collagenase. Interestingly, about rabbit ■2 carcinoma In an immunohistochemical study of Graf et al., Lab. T., 45, 587-596 (1981) showed that cathepsy at the front of tumor invasion. B is localized in normal cells, including fibroblasts and leukocytes, and is not found in cancer cells. I observed that. Using indirect immunofluorescence, Woolley et al. n: P, 5trauli, A, J, Barrett.

and A, Balci (eds), Proteinases and tum or1nvasion.

PP 97, Raven Press, New York (1980) and [n: L, S, Liotta and 1. R, Hart (eds ), The Tumor 1 invasion and metastasis, P P391, Martinus N1jhoff, the Hague (1982) localized collagenase in human primary and metastatic melanoma tissues. I compared it. Three out of five primary melanoma specimens and six out of I5 metastatic specimens tested showed anti-inflammatory properties. -Positive staining with collagenase antibody, but the results are either variable or too numerous to yield any results. I couldn't reach an argument. In some cases they are found in the connective tissue surrounding the melanoma nest. In other cases, they found fluorescence at the junction of tumor and normal tissue.

Data from our previous observations on heparanase activity in melanoma cells suggested that heparanase is localized in the cytoplasm and on the cell surface. Hebara The enzyme activity was detected in intact melanoma cells and their homogenates (Nakajima et al. et al., 5science, 220, 611-613 (1983), and It was also recovered from plasma membrane vesicles from which these cells were stripped. Other reports also indicate that heparanar suggesting that enzyme activity is plasma membrane-associated or secreted by malignant cells. Ru. Hebalanase activity was determined during serum-free conditioning of metastatic ESb mouse lymphoma cells. , and further [■5) So, -incubation with labeled subcutaneous extracellular matrix It has also been observed in intact, visible ESb cells.

Vlodavsky et al, Cancer Res, 43. 270 4-2711 (1983). In another study, 5avion et al.

J, Ce11. Physiol,, 118. 169-178 (1984) Mouse activated T lymphocytes and inflamed peritoneal macrophages have the ability to release hevaranase. reported that heparan sulfate has the ability to degrade heparan sulfate in the subendothelial extracellular matrix. Ta.

These results suggest cell surface association or cell secretion of hevaranase. On the other hand, Ricoveri and Cappelletti.

CancerRes, 46.3855-3881 (1986) is a metastatic tumor. Cellular heparanase is a lysosomal enzyme, and in vitro suggested that it would not be released. However, immunochemical localization of heparanase It has not yet been attempted.

Melanoma heparanase, or endo-β-D-glucuronidase, is a It is thought to play an important role in the metastasis of the human body (Nakajima e t al, , J, cell.

Biochem, 36. 157-167 N988), which has been It is one of several tumor cell degrading enzymes that have been systematically studied. heparanase activity As mentioned above, human and mouse melanoma cell homogenates and their It is found all over Narashinji. Anti-hevaranase can be successfully prepared. allows for the determination of mouse and human heparanase levels in normal and cancer tissues. become. A number of characterization experiments were performed to confirm the specificity of the antibody.

Immunoprecipitates of antibodies from melanoma cell lysates were analyzed for proteins recognized by antibodies. 5DS-PAGE was performed to determine the molecular weight. The pIs of immunoprecipitated proteins is also 2 -Examined using simendional gel.

In an attempt to aid in the purification of heparanase, Affi-Ge1tO ( Anti-heparanase antibody-agarose affinity assay using Bio-Rad) I also assembled a mu. Heparanase activity was eluted with citrate buffer at pH below 4. Detected only in fractions. Anti-mouse heparanase antibody crosses human heparanase The finding of cross-reactivity builds on our previous findings regarding human and mouse heparanase. consistent with observation. Human and mouse heparanase are characterized by molecular weight, substrate and inhibitor specificity. share several biochemical properties, such as optimum pH for sex and enzymatic activity. . These observations suggest that the hevaranase molecule is highly conserved between mice and humans. It suggests that there is.

The production of anti-heparanase antibodies also localizes heparanase in melanoma cells and tissues. give us the opportunity to make decisions.

Heparanase antigens are predominantly found in two cell types: B16-BL6 and A375-3M. It is localized in the cytoplasm and also on the cell surface of these cells. heparanase antigen throat fraction (cell surface, cytoplasm, or both) exhibits the active form of this enzyme This will require further research in the future. Cell surface, cytoplasm and hevarana The correlation between the secreted form of the enzyme and the regulatory mechanisms involved is clear. There isn't. Immunochemical staining of B16-BL6 and A375-5M melanoma cells are heterogeneous, which explains the knowledge of tumor cell heterogeneity in gene product expression. Doesn't contradict what you see.

Hebalanase activity is associated with the metastatic potential of the mouse 816 melanoma cell line. Tokara (Nakajima et al, J.

Biol, Chem, 259.2283-2290 (1984), Heparana The enzyme antigen was investigated in mouse and human melanoma tissues. in mouse experimental metastasis model Metastatic lung nodules were stained with anti-heparanase antibodies, but normal lung tissue surrounding the tumor was stained with anti-heparanase antibodies. Almost no background staining was observed. Background in normal lung tissue may be due to the presence of alveolar macrophages in which heparanase activity was observed. (Nakajima et al, 1.cell, Biol, 101, 215a (1985); 5avjon et al, J, Ce11. Physiol, 118° 169-178 (1984). The present invention Take advantage of the fact that the US-heparanase antibody cross-reacts with human heparanase. Utilizing Immunochemical Localization Studies of Heparanase Antigens in Human Melanoma Tissues made possible.

Melanoma tissue from patients with metastatic disease was examined.

Melanoma cells are stained with antibodies, while surrounding normal tissue is.

The endothelial cells of small arteries (Fig. 3c) were only lightly stained and not stained. endothelial cells are Expresses heparanase along with other degradative enzymes during neovascularization and wound healing processes there's a possibility that. The data submitted consistent with the present invention are in invasive melanoma cells. suggesting that the heparanase antigen in the cells is significantly increased compared to surrounding normal tissue. There is.

The localization of hebalanase antigen on the melanoma cell surface suggests that the cell surface is involved in tumor invasion and metastasis. Supporting the idea of engaging (Nicolson, Biochim, Biop hys, Acta, 695, 113-176 (1982); Mo5ca telli et al, Biochim.

Biophys, Acta, 948. 67-85 (1988), Melanor localization of hebalanase to invasive and metastatic melanoma cells in melanoma tissues. Strongly suggest that this enzyme plays an important role in melanoma invasion and metastasis. .

Such localization may lead to changes in therapeutic strategies. This is due to metastasis and invasion. More aggressive tumors appear to contain more detectable hevaranase and are more aggressive. This is because there is value in providing appropriate treatment.

Endoglycosidase according to Gerson (1984) J, Cl1n.

[mmunoassay, V7, P73-81 reviewed the fluo Quantitative methods using recein or other fluorescent compounds, Weeks and Wo odhead (1984), J, Cl1n, [mmunoassay.

V7. Chemiluminescence quantitative method reviewed in PP82-89, etc. It may also be determined by quantitative labeling methods. All of these quantitative methods Therefore, conjugated endoglycosidase can be converted to non-conjugated endoglycosidase by various methods. isolated from Dase. These methods include primary (anti-endoglycosidase) anti- Immobilization of the body onto a solid phase, avidinization using biotin-labeled antibodies and solid phase avidin. biotin separation, double antibody precipitation, or habten coupled to the primary antibody. using a solid-phase antibody against similar fluorescein, or using a solid-phase “2-charcoal body” etc. are included. (“Double antibody” refers to Midley, et al.

(1969) Acta Endocrinol, V63.5uPP, 142 ゜As in PP47, a non-corresponding antibody that binds to an anti-endoglycosidase antibody and ).

The solid phase systems mentioned above are polymers such as polystyrene, agarose:Sepharose:cell. loin; glass peas; and convertible particles of cellulose or other polymers. can include. Solid phase is large or small beads or particles; tube ; plate: or other shapes.

Kits useful in the present invention include 5zczesniak CU, S, Pat, No. 3.

899.298).

Such a kit may contain at least 11 or at least 2 or at least 3 or It has a carrier partitioned to accept more containers and is closed. Maintain the container in a restricted condition. The first container contains purified anti-glycosaminoglycans. Freeze-dry endoglycosidase antibody (preferably monoclonal) in solution. either the mold or a covalent bond to their interior - e.g. or test tubes - included. The second container contains a second anti-glycosaminoglycan. Contains endoglycosidase antibodies (again preferably monoclonal).

Otherwise, other containers contain detectably labeled glycosaminoglycan endoglycosylation. Contains Dase antigen. The glycosaminoglycan endoglycosidase antigen in the sample When testing, add the sample to the first container containing the monoclonal antibody and ink. Then add the antibody from the second container to make the “sandwich.” Prepare.

The antibody in the second container can be detected by, for example, a radioactive label or an enzyme label. There are no signs. Other containers in the kit are for carrying out the “ELrSA” method. Contains appropriate enzyme substrates. Glycosaminoglycan endoglycosidase antigen We must keep this in mind when creating kits to accommodate the various methods used to detect It will be done in a timely manner. Which one to choose depends on ease of handling, speed and Determined by test efficiency.

Quantitative analysis of glycosaminoglycan endoglycosidase antigens is well known. This is done by interpolation to the drift curve as shown in the figure. The variety of containers means that different amounts of Each container containing the glycosaminoglycan endoglycosidase antigen is This is because the purpose was to make it appear in the list.

In yet other embodiments, antibodies can be immobilized on plastic strips and then This is a sample suspected of containing glycosaminoglycan endoglycosidase antigen. bring into contact with. Then add the strip to a second, enzyme-labeled anti-glycosaminoglyceride. contact with a solution containing lycan endoglycosidase antibodies; A sandwich is formed on the top. Finally, place the strip in a coloring solution (for enzymes). This is quick, efficient, and qualitative In animal samples, glycosaminoglycan endoglycosidase It can also be a crude quantitative method for measuring enzyme antigens.

The immunoassay method of the present invention uses different glycosaminoglycan endoglycosidases, A distinct antibody for heparan sulfate endoglycosidase Use. The methodology described here is useful for glycosaminoglycan endoglycosidase assays. It must be superior in sensitivity and ease to other methods known for No.

In the same way, the kit can be used to connect the molecular beacon, preferably through the amino terminus, as described above. It is easily assembled to contain labeled glycosaminoglycans. This way The kit also contains labeled glycosaminoglycans or labeled fragments thereof. It will also include a specific binding agent that can remove it from solution. Specific binding agent is a solid matrix either already conjugated to the kit or can be conjugated by the user of the kit and assay method. It becomes. Preferred binding agents are proteins, more preferred are antibodies, and most preferred are antibodies. Preferred are monoclonal antibodies.

A unique diagnostic test for glycosaminoglycan endoglycosidase infections Its development has become a medical necessity for purposes such as tumor detection. here Such specific diagnostic tests described in Glycosaminoglycan Endoglycoside through the use of monoclonal or polyclonal antibodies that specifically bind to will be developed.

These examples are presented to illustrate the advantageous aspects and benefits of the present invention; The present invention is not limited unless otherwise stated in the appended claims in this regard. Melanoma hevaranase was purified from cultured mouse melanoma cells. Mau B16-FIO melanoma cells (Fictler, Nature (Londo) n) New Biol, 242, 148-149 (1973), Dul Becco's modified Eagle's medium and heat-inactivated 5% fetal bovine serum (FBS): Hyc 1 one, Logan.

Ham’s F-12 nutrient mixture (DME/Fl 2; Ha zleton, Lenexa, KS) in a l=1 mixture.

Cells in near-confluent culture were incubated at pH 7°2 (PB S) were collected by brief treatment with 2mM EDTA in phosphate-buffered saline, and 2X 10 ``cells were treated with 1mM phenylmethylsulfonyl fluoride (PMSF+Si gma, St.

Louis, MO), 5mM N-ethyleneimide (NEM; Sigma), 0.5% Triton X-100 and 0.05% sodium azide (buffer 1) in pH 7, 5, 50mM Tris-HCI 50-, at 4°C for 30 minutes. Extracted between. 30. Cell extract Centrifuge at 4°C for 130 minutes at OOOxg, approximately 50 Heparin-Sepharose (Pha rmacia LKB Biotechnology, Piscataway, NJ) column. Heno-Sepharose column was sequentially washed with buffer 1. 20mM vinegar at pH 6°0 containing 0.2% Triton X-100 (buffer 2) and 0.15M sodium chloride, 20mM sodium acetate, p Washed with H6,0 (buffer 3). Heparin-binding protein was dissolved in 20mM sodium acetate. linear sodium chloride gradient (0,15M-1,2M ) was eluted. The eluted material was monitored by measuring absorption at 280 nm and -ase activity was measured using heparanase solid phase substrate as previously described (Naka Jima et al., Anal, Biochem, 157.

162-171 (1986b). The hebaranase activity fraction contains approximately 6 μg of protein and total It contained 90% of the activity. After dialysis with buffer 3, the heparanase fraction was Concanavalin A was centrifuged at ,000xg for 30 minutes and the upper groove was equilibrated with buffer 3. - Loaded onto a Sepharose column (Pharmacia LKB). Loosen the column After washing with buffer 3, the concanarin-binding protein was dissolved in buffer 3 at 1.0 Mα- Elution was with methyl-D-mannopyranoside (Sigma). Approximately 0.　2■ Egg The eluate containing 48% of the white matter and total heparanase activity was collected and permeabilized thoroughly with Buffer 3. N-acetyl N-desulfated hebarin-cephalogram was analyzed and equilibrated with the same buffer. column (Nakajima et al, Anl, Bjocheffl,... 157. 162-171 (1986b). 0.3M sodium chloride After washing with 20mM sodium acetate, pH 6, the bound protein was washed with 0.6M sodium chloride. Eluted with sodium chloride, 20mM sodium acetate, pH 6.0. Highly refined hexafluoride Paranase fraction (0.08μ protein, 34% of total activity) was added to 12.5mM Tri s-MCI! , 0.15M NaCf, pH 7.5, Centricon Concentrate with a 30 concentrator (Am1con, Danvers, MA), then PR OTE INFAK300SW column (Waters, Milford, M A) High performance gel permeation chromatography using a Waters 600E system equipped with I applied it to the graph. Protein with 12.5mM Tris-HCI! , 0.15M Elute with NaCj7, pH 7,5 (1 i/min, 23°C) and transfer the eluate to Waters Monitored at 280 nm with a Model 490 multi-wavelength detector. Molecular weight approximately 100 ,000 to 150,000 (0.04 ■ protein, total activity 29% of sex) contained hevaranase. Heparanase fraction was further divided into PBE9 Chromatofocusing was performed using 4 columns (Pharmacia LKB). . Starting buffer and elution buffer were 25mM imidazole-HCl, pH 7, respectively. 5 and polybuff y-74-HC1 (Pharmacia LKB), pH 4° It was 0. Hevaranase elutes as a sharp peak at pH 5.0-5.2 Approximately 0.01 μg of protein with 18% of the total activity was recovered. refined 1<Lanase Laemi Nature (London) 227. 680- Sodium dodecyl sulfate (SDS)-polyacrylate by the method of 685 (1970) The apparent molecular weight was determined to be 97,000. Ta. Cut out the lanase band and use the TSCOSC Forensic Concentrator Model 1750. (ISCO, Lincoln, NE) and then The amino acid sequences were analyzed in a model 470A gas furnace with a model 120A-PTH analyzer. By phase sequencer (Applied Biosystem Inc.) analyzed.

immunoreagent Hebalanase bebutide (EEDLGKSREGSRTDD-C) amino acid N-terminal sequence (EVDVDGTVEEDLLGKSREGSRTDD) Merrifield J.

According to Am, Chem, Soc, 85.2149-2154 (1963) , DuPon was synthesized on a solid-phase peptide synthesizer. Peptide added to C-terminus The cysteine residues are easily conjugated to the carrier protein, keyhole Kasagai hemocyanin. (KLH; Calbiochem, La Jolla, CA) Of course, other protein carriers can be used in place of KLHO. KL Conjugation of peptides to H is performed by Liu et al.

Biochemistry, 18. 690-697 (1979) states that It was carried out using the following method. To put it simply, KLH was dissolved in 10mM sodium sulfate buffer. Suspended in pH 7.2 (PB) at a concentration of 20 μ/ml, molecular weight 12.000-14 Cellulose membrane that cuts off ゜000 (Spectrum, LosAnge) les CA) against 2 L of the same buffer overnight (replacement of fluid 3 times). The dialyzed material was centrifuged at 10,000xg for 10 minutes to remove insoluble materials. Ta. Pierce using bovine serum albumin (Sigma) as a standard.

Coomassie protein quantification method (Pierce, Rockford, IL) ), and the concentration of KLH was adjusted to 16 μ/rItl. peptide Dissolved in PB to a concentration of 5μ/rnl, m-maleimidobenzoyl-N-hydride Roxysuccinimide ester (MBS; Sigma) was added to dimethylformamide. (DMF; Aldrich, Mj1waukee, WE) 6 The concentration was mg/ml. MBS solution was converted into LHK solution (MBS: KLH= 0.51■, :4.0■) and stir the mixture gently. and incubated for 30 minutes at 25°C. activated by gel filtration chromatography The modified KLH was separated from the remaining low molecular weight MBS. That's Bi.

- Void volume of Gelp-30 gel filtration column (2,5X25an) and eluted with 50mM phosphate buffer at pH 6.0. Peptide solution (5■/rd) was mixed with activated KLH and the pH was adjusted to 7.0-7. Adjusted to 5. After incubation at 25°C for 3 hours, the peptide-KLH mixture The material was centrifuged at 10,000xg to remove insoluble material. Conjugation efficiency of free sulfur It was measured using the Elman quantitative method for , Biochem, Biophys, , 82. 70-77 (1959) Conjugate The efficiency was over 78.5%. Measure a fixed amount of KLH-conjugated peptide antigen 8 Stored at 0°C.

New Zealand white rabbits were immunized subcutaneously with KLH-conjugated heparanase peptide. . Prior to injection, the antigen was administered in either complete Freund's adjuvant for the primary immunization or subsequent Emulsified with incomplete Freund's adjuvant for enhanced immunity. the first For injections, the antigen dose was 500 μg of peptide, and for subsequent booster injections. It was 200 μg. The interval between the first and second injections is 3 weeks, and the booster injection is serum. This was done when the titer started to drop.

Antibody activity against hebalanase peptide was determined using enzyme-linked immunosorbent assay. Performed on peptide coated 96-well plate (Co5tar, Cambri dge, MA). Plates were treated with 0.1 M sodium bicarbonate, pH 9.0, for each well. io.

μ! Coat with 1 μg of synthetic peptide antigen and allow the buffer to evaporate overnight at 37°C. Ta. The titer of antiserum raised against heparanase peptide was compared with preimmune serum. When this was done, I:800 and l:1200.

Antibodies against hebaranase to peptide were prepared using Affi-Gel 10 beads (R1 Heparanase covalently conjugated to o-Rad, Richmond, CA) - peptide was further purified using antigen affinity chromatography. serum was first precipitated with 45% (V/V) ammonium sulfate at 4°C and 10.000x Centrifuged for 20 minutes at g. The precipitate was then mixed with 5mM HEPES and 150mM NaC. 1, pH 7, and 4, the buffer solution was changed three times and dialyzed overnight at 4°C. Dialysate A Loaded onto ffi-Gel-heparanase peptide affinity column. Af The affinity column was prepared with 5mM HEPES, 150mM Na (I!, pH 7,4 Wash thoroughly with 50mM sodium citrate, 150mM NaCl, I) H5,5 and finally 50mM sodium citrate, 150mM NaC 1, eluted at pH 2.0. Collect fractions and measure protein concentration using IgG as standard protein. Measured by Pierce Coomassie protein quantitative method using did. Antibody activity against heparanase peptide was measured by ELISA assay. . The antibody fraction eluted at pH 2.0 was collected and named as anti-hevaranase antibody. is. Antibodies were prepared by Updyke et al., J. Immunol, Meth, , 73.

83-95 (1984).

Heparanase peptide is alkylated with iodoacetamide to create a C- The terminal SH group was inactivated and an anti-heparanase antibody was used as a control in immunoprecipitation experiments. It was used to compete with Reaction: 2mM peptide, 1mM dithiothreitol, in 20mM iodoacetamide, 50mM Tris, 150mM sodium chloride , pH 7° and 5.25°C for 1 hour. Concentrate sample and alkylated peptides was separated on a Blo-Ge1 P-2 column (Mesh 400). Voidobori The volume was collected and lyophilized.

Cells and cell culture Human A375=M selected in nude mice for enhanced lung colonization ability M and A375-3M melanoma cell lines (Kozlowski et al. , J, Natl, cancer [nst,.

72, 913-917 (1984) by Dr. J. Fldler ( University of Texas M, D, Anderson Can cer　Center.

Huston, TX) and sequentially selected for enhanced bladder invasion. mouse B-16-BLl & melanoma cells (Hart, Am, J, Path ol,, 97.587-600 (1979) by Dr. [, R, Hart ( Imperial Cancer Re5eavchLaboratories , London, England). Pass the cells through 5% CO2 gas. 5% heat-inactivated FBS was added in a 37°C tissue culture incubator. D It was cultured in a 1:1 (V/V) mixture of ME/F12. immunochemistry cells Multi-chamber slides (Miles 5 scientific , Naperville, IL).

Immunoprecipitation and autoradiography Melanoma cells were plated onto 1O-an tissue culture dishes. Cells near confluence When this time, the medium was aspirated and the cells were placed in Dulbecco's phosphate buffered saline (DPBS). ) and then 50 μCi/mA of (“S)methionine ([CN , [rvine, CA) by adding DME/F12 and 5% dialyzed FBS. I got it. After 24 hours of metabolic labeling, the cells were washed with DPBS, containing Ca'' and Mgt-. Incubated with 2mM EDTA in PBs for 10 minutes. Then add 6 cells Incubate for 5 min at 00xg and transfer the pellet to 6 x 10 Imt’s per cell. 0.5% TritonX-100, 50mM Tris-HCll at the ratio of Digested with 1mM PMSF, 5mM NEM pH 7.5, Vortex machine Cultivation was continued at 25° C. for 10 minutes while stirring. Then trichloroacetic acid-precipitation The specific activity was obtained by measuring the radioactivity, and the protein concentration of the cell lysate was determined. specific activity was higher than 5xlO'cpm/μg protein. 200 μg anti-heparaner Incubate the cells containing the VOrteX antibody for 1 hour at 4°C, then Add streptavidin-agarose (BRL, Bethes da, MD) and incubated for 1 hour at 4°C. Sample 0.5 10mM HEPES with % Triton X-100, 150mM NaC n.

Wash once with pH 7.5, then 10mM HEPES, 150mM NaC Washed 4 times with A', pH 7.5.

Precipitate the SDS sample in the presence or absence of 2.5% β-mercaptoethanol. suspended in buffer and heated at 100"C for 3 minutes. They were then suspended in the presence of SDS. 7°5% polyacrylamide gel (Bio-Rad, Richmond , CA). The gel was then washed briefly with deionized water, dried, and then prior to development on a reinforced screen with Kodak XOMAT film. The device was kept at -80°C for 2 days. ”C-labeled molecular weight standards (Sigma) for electrophoresis included.

Mouse and human melanoma tissue Preparation of mouse metastatic melanoma tissue is as follows. 8 in a state close to densification 16-The BL6 cell layer was washed with DPBS, and Ca"" and Mg! PBS without + Incubate for 10 minutes with 2mM EDTA and remove military cells in DME/F12. Suspend as cells, cool in an ice-water bath before centrifugation, and then add 2.5X10' cells. Resuspend in cold DME/F12 at a concentration of cells/ld. Immediately before injection, cell suspension Warm to room temperature and transfer an aliquot of cells (5 x 10 cells in 0.20-medium) into a 27-gauge needle. was used to inject into the lateral tail vein of 6- to 8-week-old C57/BL6 mice. each experiment using a mouse. After 2-3 weeks, the mice were exsanguinated under anesthesia, and the lungs were carefully removed ( It was removed by cryoablation.

Human melanoma tissue was obtained from seven patients diagnosed with malignant melanoma. human mother Plaque biopsies were obtained from normal donors. Frozen tissue sections were prepared as described below. did.

Indirect Immunocytochemistry and Immunohistochemistry Multichamber Slides (Miles Melanoma cells grown on 5 scientific Fixed with luminaldehyde for 20 minutes at 25°C, then rinsed three times with PBS. go Permeabilize some cells with cold acetone for 3-10 minutes at -20°C and incubate with PBS at 25°C. I rinsed it three times. Fresh masks and human tissues were exposed to O, C, T, stump shape with 7 running double ground (Miles 5 scientific) Place it on top of the cryostat microtome (Daman/rEc ustommicrotome, NeedhamHeights, MA) Sections were made (2 μm thick). Sections were washed with 2% paraformaldehyde in PBS. It was fixed at 25°C for 20 minutes. After rinsing three times with PBS, slides were pre-immunized with rabbits. IgG for 1 hour, followed by PBS, anti-heparanase antibody or Anti-heparanase was incubated with synthetic peptide antigen for 1 hour at 25°C. and incubated with enzyme antibody. After rinsing three times with PBS, slides were placed in PBS. peroxidase-conjugated goat anti-rabbit gG (C albiochem, La Jolla, Calif.) for 1 hour.

The slide was then treated with 3-amino-9-ethylcarbazole (ABC; ma) Developed in buffer: 1.0-N.

4μ of AEC dissolved in N-dimethylformamide (Aldrich) was added to O , 1M sodium acetate, p)t! Dissolved in 5.2+1-2 drops of 30% H20z . 10g of gelatin (Fisher 5 scientific) was steamed at 60mN'. Glycerol-gelatin prepared by dissolving it in distilled water and heating it until the gelatin dissolves. Cover the slides with a coverslip using medium containing 70% of the gelatin solution. m/m of glycerin and 1 mA' of phenol were added.

Dr, N, Di Ferrante (Baylor CoCo11e of M edicine.

Houston, TX), and its average molecular weight was determined to be 34.000 by the sedimentation equilibrium method (Nakajima, M., et al. ,, (1984) J, Biol, Chem, V 259, PP2283-2 290 and Irimura, T., et at.

(1983) Anal, Biochem, Vl 30. PP461-468 ). Pig intestinal mucosal tissue was prepared by Kano S, M, B, Mathews, J, A.

C1 fonelli, and L, Roden (University of Kindly provided by Chicago, rL). Chondroitin from shark cartilage 6-sulfuric acid (C6S) is a Miles 5 scientific (Naperville [L] and further purified by gel chromatography; The average Mr. was determined to be 60,000 as mentioned earlier (Irimuva, T. , et al., (1983) Anal.

Biochem, Vl 30. PP461-468), bovine lung and pig intestinal mucosa Heparin and N-acetyl-D-glucosamine from Sig+na Chemi Cal Co, (St., Louis, MO).

Monosialosylubiantennary complex-type glycopeptide UB-I -b (Mr 2190) was prepared from thyroid globulin (Sigma) ( Irimura, T, et al.

(1983) Anal, Biochem, Vl 30. PP461-46 8), Plavobacterium heparium (E C4, 2.2°8) was purchased from Miles 5 scientific.

High performance gel permeation chromatography. As mentioned previously (Irimura, T. , et at, (1983) Anal, Biochem.

V130. PP461-468), Fractogel (Toyopearl) T S K KW -55 (s ) (MCB, Gibbstown, NJ) with two consecutive columns. using a body chromatography system (LDC, Riviera Beach, PL). High performance gel permeation chromatography was performed.

100 μl of the sample solution was placed into the injection boat and the chromatography elution was carried out at 55°C. 1.0rrd! Performed using 0.2M sodium chloride at a flow rate of /min (Ir imura, T, et al.

(1983) Anal, Biochem, Vl 30. PP461-46 8). In the analysis of radiolabeled substances, each elution (0,6mt') corresponds to 36S. Collect the fractions and apply Liquiscint (National Diagnost ics, Comerville, NJ), then Beckm an LS 2800 liquid scintillation counter (Beckman In force (Cappelletti, e tal, , Anal, Biochem, , V 99, P 31l-31 Glycosaminoglycans were analyzed by cellulose acetate electrophoresis according to method 5). analyzed.

Titanl [IZip Zone Cellulose Acetate Plate (6, OX 7, 6 an, He1ena Laboratories.

Beaumont, TX) and Cappelletti et al. Cappelletti.

et al, Anal, Biochem, V99. PP311-315 ) used 0.5M pyridine-acetic acid (pH 5.0) instead of 0.1M barium acetate. ) Electrophoresis was carried out at 70 V for 60 minutes. During electrophoresis, buffer and cellulose For the acetate plate, use petroleum ether cooled with ice, and if the temperature exceeds 4°C, use Nag. asawa and Inoue (Nagasawa, et al, (19 77) Method in Carbohyd, Chem, V8. PP 2 91-294). Sodium salt of purified H3 AC30Wx 8 (H” form, Bio-Rad, Richmond, CA) on a column, Converted to vinyldinium salt by cation exchange chromatography and neutralized with pyridine. It became sexualized. Complete N-sulfation and partial N-sulfation of HS were performed using 95% dimethyl sulfate. Phoxide and 20°C for 60 minutes, respectively. The pH of the reaction mixture was 0. IM Add NaOH to adjust the temperature to 9.0, mix the mixture with tap water overnight, and then with distilled water. Dialysis was performed for 20 hours. N-acetylation of N-desulfated HS (4% acetic anhydride, Run for 3 hours at 4°C in 4M sodium acetate, pH 8.0, containing 15% methanol. provided. Dialyze the reaction mixture overnight against tap water and distilled water, then freeze-dry the mixture. susceptibility to melanoma heparanase (=in order to examine the effect of HS was labeled with IJ tium (at the reducing end) as described in (Nakajim et al. a. M. , et at. , (　1　98　4)　J. Biol. Chem. V2 5 9. PP2 2 8 3-2 2 90). Purified HS 1■ is heated to O, 2 mC i in 1M sodium borate buffer, pH 8.0, 5 hours at 25°C. of NaB ('H) 4 (3 4 0mC i/mmo 1; NewEn grand Nuclear. Boston. MA). pH5 with acetic acid After acidification to 0.2 M pyridine-acetate buffer, pH 5. A column of Sephacryl S-200 equilibrated with 0 (1. Chromatography was performed on 0.5 ann). 3H-labeled HS with specific Mr The images were collected and freeze-dried. Solid-phase henolanase substrate (combining two radiolabeled HS) The partially N-desulfated HS was treated with (1 = “C) acetic anhydride for Cetylated. 15μ of partially N-desulfated HS was added to 4M sodium acetate. , 0.15 mC+ (1-I4C) acetic anhydride (10, O mC+/mmol; New England Nuclear) and 4°C Incubated for 4 hours at 4°C in the same buffer. Incubated. The reaction mixture was extensively dialyzed against distilled water and then lyophilized.

As mentioned above, partially N-sulfated N(]4C)acetylated HS (PNDS- N (“C) Ac-HS) high molecular weight fraction) Sephacryl S-2 Obtained by gel chromatography on a 0.0 column.

"Conjugate of 14C-marked HS to pu."C - Reductive amination of the reductive terminal saccharide of labeled HS is as follows.

Dissolve PNDS-N M’CI Ac-HS (5mg) in 5rIL of distilled water. 4M ammonium formate and O in methanol.

Mix with 5rd of 8M Sodium Cyanoborono\Idlide, then heat at 50″C for 7 Incubated for days. The reaction mixture was dialyzed against distilled water and freeze-dried. reductive The product aminated to 0. IN Sodium Bicarbonate, 10- dissolved at pH 8.5 2. By sequentially washing with propatool and then with ice-cold distilled water. Affi-Gel 15 (or Affi-Gel 10; Bio-Red). Gently mix the mixture and heat at 4°C. Incubated for 24 hours. Then 0. IN sodium bicarbonate to pH 8.5 Adjustments were made and further incubation was continued. 24 hours later Affi-Gel 1 Block the unreacted position of 5 by adding 1M glycine ethyl ester (pH 8.0). and the beads were incubated again for 6 hours at 4°C. After the reaction is complete, the conjugate The product was thoroughly washed with 1.5M sodium chloride, 0.1M sodium acetate, and 0.1M sodium chloride. 15M Sodium Chloride, 0.2% Triton X-too and 0.

Incubate overnight at 37°C in 0.5% sodium azide (pH 6.0). Ta. The product was further washed in the same buffer and stored at 4°C.

A summary of the synthesis method described above is shown in FIG.

Hikime Σ The 2L Koi' (Higoyu Fudanyuu' Libi') lization and conjugation to agarose beads . +40- or “H-labeled HS was chemically deacetylated and irradiated as shown below. prepared by reacetylation. Bovine lung HS (Dr. N. DiFerrant e, Baylor CoCo11e of Medicine, Houston , TX) over phosphorus pentoxide with 1 part of hydrazine sulfate under vacuum at 50°C. It was dried for 48 hours. Anhydrous hydrazine (0,5+og, Pierce Chemical, Rockford, IL) and screw the mixture tightly. The mixture was heated at 100° C. for 1 hour in a nitrogen atmosphere in a tube closed with water. After reaction, add sulfuric acid in vacuum Hydrazine was removed by repeated evaporation with toluene on a desiccant. . Completely dry to separate the deacetylated H3 from residual reagents and partial degradation products. Dissolve the residue in 0.5-g water and add it to 0.8 x 30 cm BioGel p-10 ( Gel filtration was performed on a particle size of 400) and eluted with distilled water. Collect void volume fractions It was then lyophilized. The yield was approximately 60% by weight. Next, N-hypothesis acetylation HS 50 μCi (I4C) acetic anhydride (10 mCi/mmo 1e: NEN , Boston, Ma) or 5mC1'H-acetic anhydride (400mCi/mmo 1e:NEN) for 18 hours in 0.5-4M sodium acetate. Acetylation was performed. Complete acetylation was mixed with unlabeled acetic anhydride for 1 hour. and completed it. As described above, 14C- or 2H-labeled H3 was added to the same BioG Purified on elp-10 column.

For solid-phase hevaranase quantification, 0° 4M sodium cyanoporin in 50% methanol ``H-H3'' at the reducing end position with 2M ammonium acetate in the presence of hydride. Amination was carried out for 6 days at 50"C. Aminated sH-H3 was purified by gel filtration as described above. The resulting solution was purified and the resulting solution was dissolved in sodium bicarbonate. I changed it to IM.

After washing the gel beads with isopropanol and cold water according to the manufacturer's recommendations, cpm amination “Add 1.0-Affi-Gel 15 to H-H3 did. The military reaction was continued for 48 hours at 4°C with constant stirring. Then agarose The beads were washed repeatedly with 4M sodium chloride to remove non-covalently bound "H-HS" from the beads. removed from the list.

Melanoma cells and cell culture. Metastatic mouse B16 melanoma with high lung colonization potential -ma subline (B16-Flo) and 14 established human metastatic melanoma cells strain was used in this study. The human melanoma cell lines used are as follows: SK-MEL-19, SK-MEL-23, SK-MEL-93 (DXI), SK-MEL-93 (DX3), SK-MEL-93 (DX6), H329 4T, HS852TSHS939, T294, M2O, RON, BMCL, A3 75 parent strain and A375Met Mixa A375Met Mix1M cells are free. Obtained from natural lung metastases of A375 parental cells in thymic mice, both A375 The cell line is Dr. 1. J, Fiddle (The University of Texas-M, D, Anderson Ho5pital and Tumo Provided by In5titude, Houston, TX). Melanor The cells were grown on plastic tissue culture dishes, which were modified by Dulbecco. modified minimum essential medium and Ham's F12 medium (DMEM/F12; Gibco L A 1:1 mixture of Aboratories, Grand Land, NY) This mixture contains no antibodies and contains 10% fetal bovine serum (Hyclon, 5terile Systems, Inc.

Logan, UT), placed in a 95% air-C02 atmosphere under humid conditions. There is. All cell culture fluids were tested using mycoplasma detection system (BRL Myco-Tes). t; Bethesda Re5search Laboratories, Gai Thersburg, MD) to confirm the absence of mycoplasma contamination. Ta.

Melanoma cells close to yifR0 confluence of cell extracts with Ca2+ and Mg! “Nona treated with 2 m diaminetetraacetic acid (EDTA) in 2 m DPBS for 10 min. Collected. Military-cell suspension in 0.14M sodium chloride, 10mM Tris-H Wash twice by brief centrifugation in CI buffer, pH 7.5 to exclude trypan blue dye. The survival rate (usually 95% or more) was checked using a method. Cells 6XI O@cells/- Cold 50mM Tris-HC containing 0.5% Triton X-100 at a concentration of Suspend in f buffer, pH 7.5 and extract for 5 min at 25 °C and for a further 1 h. did.

After centrifugation at 9800xg for 5 minutes at 4°C, the supernatant was collected. The protein concentration in the centrifuged extract was Lowry method to correct for the presence of Triton X-100 in the sample. Modified method (Nakajjma, M., et al., (1984) J. Bjol. Chem, V 259, PP2283-2290).

5M sodium chloride, 0.2% Triton X-100 and 0.05% azide 0.1 M sodium acetate (pH 6,0) containing sodium chloride (reaction buffer A)2 00μ! and 20mM D-sugar acid 1,4-lactone (SAL, a potent lucuronidase inhibitor). Incubation is Perform the reaction at 37°C without mixing gently, and cool to 4°C to stop the reaction. This was done by adding trichloroacetic acid at a concentration of 5%.

Centrifuge at 9800Xg for 5 minutes to obtain the upper groove, which is then subjected to high performance gel permeation chromatography. It was analyzed using

Heparanase quantification method using solid phase substrate. PN conjugated to Affi-Gel 15 A suspension of DS-N(14C)Ac-H8 was mixed with melanoma cell extract, 400μ of reaction buffer containing 0mM SAL! incubated inside. oxygen reaction was cooled to 4°C and stopped by mixing with 40 μl of 50% trichloroacetic acid. 4℃ After incubating for 10 minutes, the mixture was centrifuged at 9800 I got it. Add a certain amount of 100μl of the upper groove to 1.0N NaOH and Liquiscint ( Mix with National Diagnostic) 4-, Beckman L Counts were performed on an S2800 liquid scintillation counter.

N-desulfation and N-acetylation of H3 result in H3 degradation by melanoma heparanase. impact on. Using coupling reagents that can significantly change the molecular structure of HS. In order to label purified Is with a radioactive molecule without using an N-sulfate group, (“C) was substituted with an acetyl group.The basis of this idea was B16 melanoma Maheparanase has high activity against various HS molecules, but is anti-heparin. Also, the N-acetin content of H3 is high and the N-sulfuric acid content is low. This is our previous observation that heparin is different from heparin.

N-desulfation and N-acetic acid on H3 susceptibility to melanoma heparanase The effect of chilling was evaluated using H3 labeled with tritium at the reducing terminal saccharide position. I valued it. H3 purified from bovine lung mostly contains N-acetyl-D-glucoat at the reducing end. Because Samin exists (Nakajima, M.

et al., (1984) J. Biol. Chem. V259. PP22 83-2290), HS is reduced with NaB("H)a.

N-desulfurization of H3 with tritium-labeled reducing terminals by solvolysis with DMSO The oxidized and then N-desulfated ('H)H3 was N-acylated with acetic anhydride. child These three H-labeled, chemically modified HS molecules were added to 0.5 M pyridine-acetate buffer. Cellulose acetate electrophoresis was performed at pH 5.0. Described in Materials and Methods HSN-desulfated HS (NDS-H3) and N-desulfated N- The relative mobility of acetylated HS (NDS-NAc-H3) is 3.30, respectively. ．． They were 2.55 and 2°90. These findings are due to the desulfation of E(S). This shows a large loss of negative charge; however, due to acetylation of free amino groups, The total negative charge is partially recovered.

The average molecular size of NDS-H3 and NDS-NAc-H3 was determined by high-performance gel permeation chromatography. They were determined to be 30,000 and 31,500, respectively, using the matograph method ( Figure 2).

SAL each of HS, NDS-H3 and NDS and NDS-NAc-H3 816 melanoma cells in the presence of (a potent exo-β-glucuronidase inhibitor) extract and the products are separated by high-performance gel permeation chromatography. analyzed. All of these substrates were cleaved at high rates with melanoma heparanase. , the Mr of the generated fragments was characteristic for each substrate, but their elution characteristics were the same. The results were the same (Figure 2). The decomposition of each chemically modified H3 was performed in bovine lung or pig intestinal viscosity. It was completely inhibited by the addition of heparin purified from the membrane. This result shows that N-sulfur in H8 Acid is not important for melanoma heparanase recognition and cleavage, and H Chemical modification of the sulfated amino group in S significantly affects its degradation by heparanase. It shows that there is no.

N-desulfated and N-acetylated heparin. From the known structures of H3 and heparin, N-desulfation and subsequent N-acetylation of heparin are similar to the local structure in H3. It is suggested that this method creates a structure with a similar structure. Heparin is 816 melanoma heparanase (Nakajima).

M., et al., (1984) J. Biol. Chem. V259.　 PP 2283-2290) Lost when removed (Irimura, et al.

(1985) J, Ce11. Biochem, V9A, P 148).

From the above results, the N-sulfuric acid in H8 is unnecessary for cleavage by melanoma hevaranase. Therefore, N-desulfated N-acetylated heparin was used as a hevaranase substrate. I used it.

Porcine intestinal mucosa (Mr 11.000) pre-tritiated at the reducing end Heparin obtained from ) (Nakajima, M.).

et al., (1984) J. Biol. Chem. V259. PP22 83-2290) was desulfated and then used in the preparation of NDS-NAc-H3. N-acetylation was carried out by the method. The product, N-desulfated N-acetylated heparin (NDS-NAc-heparin) is a molecular A bifurcated 0° 2M pyridine-acetate buffer, pH 5, with a volume of approximately 10,500 , O, the relative electrophoretic mobility on cellulose acetate is 2H-labeled heparin. It was 0.87 when it was set as 1.0. 'H-labeled Sovalin H-ff identification ND S-NAc-heparin was incubated with B16 cell extract and the reaction product were analyzed using high performance gel permeation chromatography. Before the initial substrate degradation percentage As mentioned above, it was calculated from the decrease in the region of half the high molecular weight of the substrate peak (Na Kajima, M., etal, (1984) J. Biol, Ch. em, V259. 2283-2290). First 6 hours with B16 cell extract During incubation, up to 5% of heparin is degraded, while ND5-N Approximately 20% of the AC-heparin was fragmented. However, NDS-NAc- Heparin is not further cleaved and ND on high performance gel permeation chromatography The main peak of S-NA c-heparin remains low even after longer incubations. There was no shift towards molecular weight.

This is due to N-desulfation and subsequent N-acetylation of heparin. Heparanase-sensitive glucuronosyl conjugates are formed and/or or exposure. Therefore, NDS-NAc-heparin is effective for melanoma. A method for synthesizing solid-phase substrates for heparanase that can be used as a substrate for maheparanase quantification. It is shown in Figure 1.

To minimize the effect of radiolabel on the structure of HS, HS was dissolved in 95% DMS. Partial N-desulfation was carried out for 1 h at 20 °C by solvolysis in O and 5% H, O. It became.

As mentioned in the Materials and Methods section, partially N-desulfated HS was prepared as [1-”C] Acetylated with acetic anhydride. The remaining free amino groups are completely acetylated with acetic anhydride. did.

From these steps, PNDS-N ("C) with radioactivity of 294 cpm/μg Ac-H3 (Mr 33.OOO) was generated. cellulose acetate electrophoresis The relative mobility of PNDS-N(”C)Ac-H3 on the dynamic motion is 3.15, which is As expected, the total negative charge of PNDS-N (”C)Ac-H3O is It shows that the negative charge is closer to that of unmodified H3 than that of Ac-HS.

The reducing terminal saccharide of PNDS-N (“C) Ac-H3 is 50% ethanol Reducing with 2M ammonium formate and 0°4M sodium cyanoborohydride in is aminated to Products with free amino groups only at the reducing end are then Af Amino activated agarose such as fi-Get 10 or Affi-Gel 15 Conjugates to Subies. Backed with PNDS-N (”C)Ac-HS 5mg By incubation with Affi-Gel 15 0.5-10.2% P NDS-N (14C) Ac-HS is fixed on top of Affi-Gel 15 (0.51μ PNDS-N (I4C)Ac-H3 per 0.5-gel), P NDS-N ("C3Ac-H5 concentration 0.5-Affi-Gel 15 There was no significant effect on the conjugation efficiency under the conditions used even when the conjugation efficiency was increased to 10[ .

Conjugation of PNDS-N(”C)Ac-H3 to Affi-Gel 15 was used. Under the same conditions as PNDS-N(14C)Ac-HS and Affi-Gel 10 conjugated to. However, the conjugation efficiency was as low as 1% or less between 7.5 and 8.5.

Therefore, the positively charged spacer at the amino active position of Affi-Gel 15 is Effective sharing of PNDS-N (I4C) Ac-HS to f 1-Ge 1s Seems important to the role. Best heparanase determination using Affi-Gel 15 One of the quantitative substrates was created: PNDS-N("C)Ac-H3 was fused at the reducing end. It is immobilized on agarose by only one coexisting bond.

PNDS-N-(”C)Ac-HS enzyme immobilized on agarose gel beads decomposition. PNDS-N fixed on agarose gel (eye C) HS of Ac-HS Sensitivity to degrading enzymes was determined using substrate (4500 cpm, 15 μg) and concentration of bacterial hevaranase (EC, 4°2.2.8) containing 1mM calcium acetate. Incubated in 0.1 M sodium acetate buffer at pH 7.0. (Linker, et al, (1972) Methods Enzymo l, V28. PP902-911), after 24 hours of incubation, the majority 140 activity (82%) was found in the supernatant of the incubation mixture; PNDS-N (14C)Ac-H3 immobilized on agarose is a H3-degrading enzyme. This shows that they are very sensitive to the elements. PNDS-N (14C)A The remainder of c-HS was not released from the gel even after long incubation.

This is due to restrictions on the use of Flavobacterium heparanase. It may be possible to explain. To prevent continuous degradation by exoglycosidases, The same amount of substrate (45,000 cpm) was added to BIB cells in the presence of 20mM SAL. cells extract for various times. Amount of cell extract added (μg The correlation between protein) and free I40 activity is shown in Figure 3. In this case, free I4C activity The maximum amount of activity was 56% of the total I40 activity present in the solid phase substrate. +40 activity A part of it could not be liberated by melanoma hebalanase: Incubation of 816 cell extracts with HS or chemically modified HS Therefore, a large fragment containing the original reducing end is generated (Fig. 2). A linear relationship between the amount of cell extract added and free 14C activity was observed for each incubation. (Figure 3). obtained from 12 hour incubation. Since the obtained results are linear over the maximum range for the amount of cell extract, 12 PNDS-N (“C3Ac-HB”) degraded during the time incubation. Established.

The effect of heparin on the degradation of solid-phase substrates was determined using an equivalent amount (15 μg) of porcine intestinal tract as the substrate. Heparin from mucosa or bovine lung was added to B16-FIO cell extract (80 μg protein). PNDS-N fixed on agarose C) Ink containing Ac-H8 by adding it to the fermentation mixture. Add any heparin also inhibited the degradation of solid phase substrates by up to 80%, which is consistent with our previous results. (Nakajima, M., et al. (1984) J. Biol.

Chem, V259. PP2283-2290).

Using PNDS-N (14C)Ac-H3 immobilized on agarose Measurement of hebalanase activity in human melanoma cells. solidify on agarose beads. Using the determined PNDS-NC”C)Ac-HS, 15 human melanoma The heparanase activity of the cell lines SK-MEL-19 and SK- MEL-23, SK-MEL-93 (DX 1), SK-MEL-93 (DX 3), SK-MEL-93 (DX6), HS294T.

H3852T, HS939, T294, H40, RON.

BMCLSA375 parent strain, A375Met Mix and A375M6゜Table 1 The ability of all human melanoma cells to degrade HS in the presence of SAL as shown showed that. Six of these human malignant melanoma cell lines, namely SK-ME L-93 (DXI), SK-MEL-93 (DX6), H3939, H40 , A375Me tMix and A375M6 are mouse B16 melanoma sabra It had a significantly greater ability to degrade H3 than InFIO.

Interestingly, A375Met Mix and A375M6 cells are athymic nude tumors. It was selected from the A375 parent strain for its ability to establish lung colonies in mice. A37 5 These cells also have high metastatic potential, whereas the parental cells have very low metastatic potential. (Kozlowski et al. (1984) J, Na tl, Cancer In5t, V72. PP913-917). Therefore Thus, the hebalanase activity of A375 cells correlates with their natural lung metastatic potential.

We previously demonstrated that intact B16 melanoma from a high lung colonization capacity supply line. B16 cells or B cell extracts have a higher rate of colonization than B16 cells, which have a lower lung colony forming ability Decomposing purified H3 (Nakajima, et al. (1983) S scienceV220. PP611-613), and B16 melanoma H3 degradation Endoglycosidase is endo-β-glucuronidase (heparanase) We found that (Nakajjma, M., et al., (1984) J. Biol, Chem, V259. PP2283-2290).

Table 1 Heparan sulfate degrading activity a in human malignant melanoma cells Heparanase quantification is Tr i’ton X-100 cell extract (2,4X10’ cells) with agarose beads PNDS-N (I4C) Ac-HS immobilized on top and incubation at 37°C for 12 hours. It was implemented by Experimental details are provided in the Materials and Methods section. heat inactivated enzyme The radioactivity released in the presence of was subtracted from the raw data.

b A375 parental cells obtained from lung metastasis of A375 parental cells in athymic mice-Mix and A375M6 cells had high spontaneous lung metastatic potential, whereas A375 parental cells It had a very low spontaneous metastatic potential.

Heparanase activity in serum collected from tumor-bearing hosts was allowed to clot for 1 hour at 2°C. . Samples were centrifuged at 800xg for 10 minutes and 1600xg for 15 minutes at 4°C. profit The collected serum is divided into small fixed amounts, flash frozen in liquid nitrogen, and then stored until analysis. Stored at -80°C.

Quantification of serum for heparanase. Serum was diluted with 0.0.0 mg containing 0.15 M sodium chloride. Diluted 5 times with 1M sodium acetate buffer, pH 6.0. diluted serum 200 μ! of the radiodetection solid substrate suspension (3,000 cpm, 10 Mg) in the same buffer. 200 μf and incubated at 37°C. various incubations The enzymatic reaction was stopped by cooling to 4 °C for an hour, and the reaction sample was diluted with 50% trichloroacetic acid. mixed with. Incubate the mixture at 4°C for 10 minutes) 1,9800×g for 5 minutes Centrifuged. Take exactly 200 μf from the supernatant and add 1. ON NaOH55μ! in neutralize, then Liquiscint (National Diagnostic ) Mixed with 4-. Beckman LS 2800 liquid scintillator radioactivity Measured using a 3-sin counter. There is no direct relationship between the incubation time and the enzymatic reaction. There was a linear relationship. The activity was expressed in units per liter of serum. The activity of the 1st rank is in 1 minute This corresponds to the amount of enzyme that releases 1°Cg of HS.

Heparanase activity in serum collected from malignant melanoma patients. various disease stages Quantification of heparanase in serum collected from 20 melanoma patients and 15 normal adults The results are shown in Figure 4. Heparaner in serum from melanoma patients and normal adults The mean value and standard deviation of enzyme activity are 0.0177±0.0075 and 0.009, respectively. It was 6±0,0025 U/d. From a stupid patient undergoing chemotherapy treatment The serum heparanase activity of the patients showed normal values.

Hebalanase activity in serum collected from rats with 13762NF mammary adenocarcinoma.

F34 with highly metastatic breast cancer MTL n3 cells (IXIO”) of the same age The drug was injected subcutaneously into the left groin of female rats of the 4th strain, in the thick area of milk fat. At various times after injection Rats were sacrificed and the number of lung metastases and serum heparanase activity were determined. Sulfuric acid in serum Heparan-degrading activity increased with time after subcutaneous inoculation of MTLn3 cells (Fig. 5 ). The activity in serum correlated with the size of the primary tumor (correlation coefficient r = 0.770, Fig. 6). Does serum from rats with multiple metastases in lymph nodes and lungs cause fewer lung metastases? It showed much higher heparanase activity than rat serum without any serum (Figure 7).

Purification of melanoma heparanase. Melanoma cells (mouse 816 melanoma sub Line B16-BL6 or human melanoma Hs939 cells) were heat inactivated at 5%. The cells were grown in a 1:1 DME/F12 mixed medium supplemented with fetal bovine serum. crowding Cells close to completion were harvested by treatment with 2mM EDTA in PBS for 10 minutes, and 0.14M Washed twice in NaCl, 10mM Tris-HCI buffer, pH 7.2 . The following steps were performed at 4°C.

Cells (2X10”) in 0.2% Triton X-100, 10MM DMSF 50mM Tr i 5-HC1 containing (buffer A)! In buffer solution 30-, pH 7, 2 extracted 11111ff. 30,00 sIQg protein/-) Concanavalin A-Sepha was collected after centrifugation at 0xg for 30 minutes and equilibrated with buffer A. Loaded onto a column of Rose 4B (2X10an). After washing with buffer A of 10- The absorbed material was eluted with buffer IM α-methyl-〇-mannoside. eluate 50mM containing 0.15M sodium chloride 0.2% Triton X-100 Heparin-Sepharose CL equilibrated with Tris-HCE buffer, pH 7.2 -6B column (2X10an).

Column with 100mM of the same buffer and 50mM Tris 0.15M sodium chloride. - Wash with 100 ml of HCI, pH 7.2, then remove heparin-binding protein. Elution was performed with a salt gradient (0,15M-1,2M sodium chloride). hepara Collect the enzyme activity fractions and add 0.15M sodium chloride and 0.01M potassium phosphate. Dialysis was performed at pH 6.0. After centrifuging at 50,000xg for 30 minutes, adjust the upper groove to 0°15M. Hydroxyl equilibrated with potassium chloride and 0.01M potassium phosphate, pH 6.0 Loaded onto an apatite column (1,5x45an). Heparanase 0.15 of potassium phosphate (0.01M to 0.6M) in M sodium chloride, pH 6.0. It eluted with a linear gradient. Ultrafiltration of hevaranase fraction using YM-10 membrane It was concentrated by filtration and further purified by Sepharose CL-6B gel filtration. 0゜15M salt Sepharose CL-6B in sodium chloride and 20mM potassium phosphate, pH 6°0. Chromatographic methods were performed. Then, using Sephadex G-150, 0.5 Cloth once again in M sodium chloride and 25mM Tris-HCI, pH 7.5. A matograph method was performed. Military obtained from Sephadex chromatography - Hebarana -zebeak contained a glycoprotein of Mr96,000.

Properties of human melanoma heparanase. Melanoma hevaranase pH 5, 5 and 7 ．． 5, which degrades heparan sulfate but not other glycosaminoglycans. does not disassemble. Heparin and dextran sulfate are powerful melanoma hevaranase inhibitors It is a strong inhibitor.

! ”− Chemically modified heparan sulfate and ~(phosphorus) hebalanase susceptibility and ~lanase inhibition. Harmful activity 1, H-labeled glycosaminoglycan in 0.15M NaCl, 0.2%T 0.1M sodium acetate containing riton X-100 and 0.05% N a Fs of 2om MD-sugar acid 1,4-lactone (SAL) in um buffer (pH 6,0). Incubate with cell extract (80 μg protein) for 6 h at 37°C in the presence of This was followed by high performance gel permeation chromatography. Percentage of degradation is glycosaminos It was determined by measuring the decrease in the area of half the high molecular weight of the glycan beak (see Figure 2). + , 80% or more: ±, 5% to 15%; −15% or less (standard deviation less than 5%).

2. Incubation mixture of 2H-labeled HS obtained from bovine lung and cell extract 5 μg of unlabeled glycosaminoglycan was added. The inhibition rate of HS decomposition is determined by the HS peak It was determined by measuring the decrease in area of half the high molecular weight. ++, 88% or more inhibition; +, 25 −50% inhibition; ±, 5-25% inhibition; −15% or less inhibition (standard deviation less than 5%).

Human and mouse heparanase are antigenically related to mouse melanoma heparanase An anti-hevaranase antibody directed against the amino terminus of the enzyme was prepared as described above and then and some of the antibodies were biotinylated. In immunoprecipitation experiments, the metabolic (“S”) Thionin-labeled human A-375-3M (Fig. 9a”d) and mouse Bl 6- BL6 (Figures 9e-h) Melanoma cell lysates were first treated with biotinylated anti-Hebala. enzyme antibody (Fig. 9b, c, f, g), then streptavidin-agarose Incubated with.

Antibodies were tested against mouse BI8-BL6 (Fig. 9f, g) and human A375-3M (Fig. 9b, c). ) Major Mr~97,000 proteins or respectively from melanoma cell lysates A doublet of Mr~97.000 proteins was specifically immunoprecipitated. 5DS-P The protein properties of AC;E were the same under non-reducing and reducing conditions.

The specificity of anti-heparanase antibodies is shown in FIG.

In the presence of excess amount of competing heparanase N-terminal peptide, Mr~97,000 No band was seen (Fig. 9a, e), Mr~205. 000. ~I 2 5.　000゜～76.000. ~52,000 and 46,000 proteins are non-specifically precipitated or strebtoaviden-agarose contaminants. They appeared to be connected (Fig. 9d, h). In addition to these nonspecific protein bands , Mr ~105. 000 weak band (Fig. 9b,c.

e, f) appeared to be associated with anti-heparanase antibody precipitation; It was not inhibited by excess amount of heparanase bebutide (Fig. 9 a r e ). death Therefore, it was considered a non-specific component.

The antibody targets human proteins of Mr~97,000 from A3753M cell lysates and B1 6-Precipitated just like proteins from BL6 mouse melanoma cell lysates I let it happen. Therefore, human and mouse heparanase molecules have the same molecular weight and are antigenic. related to. To confirm the specificity of antibodies against heparanase, two-dimensional The immunoprecipitates on the gel were examined. The pI of the sediment is the value of purified heparanase, 5.0. -5.2 was the same.

Heparanase molecules have been immunochemically localized on the melanoma cell surface and in the cytoplasm. Ru Acetone-permeabilized human A375-SM (Figure 10a) and mouse B16-BL6 media. Ranoma cells are strongly stained with anti-hevaranase antibodies in indirect immunochemical quantitation. did.

Immunocytization of A375-3M and B16-BL6 cells using anti-heparanase antibodies The chemical staining appeared to be heterogeneous between cells (Fig. 10a,b). Antibodies on the cell surface Non-permeabilized A375SM (Fig. 10b) and B16-BL8, which can only react with antigen. The same permeabilized cells were also stained, although not as strongly. B16-BL6 thin The cysts stained essentially the same as the A3755M, 111 [1 cells, but Ranin deposition sometimes interfered with immunoprecipitates. Melanoma treatment with anti-heparanase Contrary to the strong staining of the vesicles, there was no detectable precipitate there. anti-he In contrast to the strong staining of melanoma cells by paranase antibodies, anti-heparanase Including when the antibody was incubated with the N-terminal heparanase peptide. There was no detectable immunocytochemical staining for any of the samples (Figure 10 d).

Mouse melanoma cells were stained with anti-heparanase antibodies in experimental metastatic tumors. Ru Hevarana in experimental mouse metastatic melanoma tumors using immunohistochemical methods position has been determined. Frozen sections of mouse lung tissue containing micrometastatic melanoma nodules It was specifically stained with anti-heparanase antibody. Test results showed that heparanase antigen was normal. It was shown to be localized in the tumor rather than in the tissue (Figure Iff). In Figures 11d-f The sections shown were taken from different levels of the same micrometastatic tumor nodule.

Serial sections show the morphology of micrometastatic foci in the upper right corner of the micrograph (Fig. 11d) . Micrometastatic melanoma nodules appear to contain large amounts of melanin and are The vesicles are heterogeneous in shape and appear to have larger nuclei than the nearby alveolar epithelial cells. . Micrometastatic melanoma nodules are strongly stained with anti-heparanase antibodies, indicating that they are metastatic melanoma nodules. The staining intensity in the lung tissue was higher than that in the nearby lung tissue (Fig. 11f). figure The same set of controls as shown in Figure 2 was also performed, but only a representative control is shown (Figure 1 ie).

No staining was seen in any of the controls, making the presence of melanin in the melanoma nodule more obvious. It became

Metastatic and invasive melanoma cells are affected by anti-heparalysis in human metastatic melanoma tissue. Human melanomas from 7 patients with metastatic melanoma stained with enzyme antibody. The presence of heparanase was investigated using immunohistochemical methods in tumor tissues. . Melanoma cells were stained with anti-hebalanase antibody, but the cells, including connective tissue, were The surrounding tissue was also essentially unstained. A typical case is skin care. Presenting ranoma (Fig. 11a-C). Malignant melanoma that invades the connective tissue of the skin Cells can be seen next to the arterioles and remnants of collagen fibers (Fig. 11a); In this case, the tumor cells lack melanin and are heterogeneous in shape. stain melanoma cells Anti-heparanase antibodies detect normal surrounding tissue, except for mild staining of endothelial cells in blood vessels. No parenchymal cells were stained (Fig. 11c). For the same control shown in Figure 10 The experiment was carried out, but only a representative one is shown in the figure (Fig, 1lb). Furthermore, human The presence of hebaranase antigen was also determined for nevi using the same method as for melanoma tissue. I looked it up. Melanocytes in normal skin in and near the nevus are anti-heparanase Not stained with antibody.

Provided herein without departing from the spirit and scope of the claims as defined below. modify the operation, operation and arrangement of the various parts, components, processes and methods described; You may.

Retention time (min) a b c d e f g h m, w, (kDa) IG S FI[G,,lQa　FTG,]1QhFIIG,,　ILQc　FIIG,- ]IQctlFI[G,,ILQe FIG-ILQffFI[G-11ILa FI[G, -11LaflFIG,]L'lh FI[G-]L]Le required Book of terms Antibodies against N-terminal hevaranase are generated. These antibodies are human and Used for the detection of heparan sulfate endoglycosidase in mouse tumors.

The localization and amount of these heparanase antigens ultimately determine the appropriate course of treatment. It will be used for planning.

Copy and translation of amendment) Submission (Article 84-8 of the Patent Act) November 1992 30-1 Shizuku

Claims (32)

[Claims] 1. Human products that appear to contain glycosaminoglycan endoglycosidase A small amount that specifically binds to glycosaminoglycan endoglycosidase to biological samples. and one antibody, and the binding of the above antibody to the sample is inhibited by glycosaminoglycans. The glycosidase assay consists of measuring the amount and localization of can-endoglycosidase. Immunoassay method for detecting the presence of lycosaminoglycan endoglycosidases. 2. 2. The method of claim 1, wherein the antibody is polyclonal. 3. The antibody corresponds to the N-terminal region of glycosaminoglycan endoglycosidase. 2. The method of claim 1. 4. the immunoassay is a radioimmunoassay, an enzyme immunoassay, or a fluorometric assay; The method of claim 1. 5. Claim wherein the glycosaminoglycan endoglycosidase is heparanase. Method 1. 6. Buffered water for human biological samples suspected of containing heparan sulfate endoglycosidase the solution and at least one antibody directed against heparan sulfate endoglycosidase. and determine the amount of binding of the antibody or antibodies to the sample using heparin sulfate. sulfate, consisting of measuring it as an indication of the presence of randoglycosidase. Immunoassay method for detecting the presence of paran endoglycosidase. 7. 7. The method of claim 6, wherein the antibody is polyclonal. 8. 7. The method of claim 6, wherein the heparan sulfate endoglycosidase is heparanase. . 9. The immunoassay is radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay. 7. The method of claim 6. 10. A kit useful for detecting glycosaminoglycan endoglycosidase in samples. At: compartmented to receive one or more container means in a tightly packed condition; the carrier on which the painting was made; A first container containing an antibody specific for glycosaminoglycan endoglycosidase. means; A detectable compound that specifically binds to glycosaminoglycan endoglycosidase. A kit comprising a second container means containing an antibody labeled with a sea urchin. 11. A kit useful for detecting glycosaminoglycan endoglycosidase in samples. At: compartmented to receive one or more container means in a tightly packed condition; the carrier on which the painting was made; No. 1 containing an antibody that specifically binds to glycosaminoglycan endoglycosidase Eye container means; detectably labeled glycosaminoglycan endoglyco A kit comprising a second container means containing a sidase. 12. wherein said antibody in said first container means is immobilized on said container means; The kit according to claim 10 or 11. 13. The antibody in the second container is labeled with a radiolabel, enzyme label, biotin, or fluorescent label. 11. The kit of claim 10, wherein the kit is labeled with a chromophore. 14. The first container means or the second container means is a polyclonal antibody. 11. The kit of claim 10. 15. Many with different amounts of glycosaminoglycan endoglycosidase antigens 12. A kit according to claim 10 or 11, comprising container means for. 16. Claim wherein the first container content or the second container content is a tube. 10 or 11 kits. 17. Claim that the glycosaminoglycan endoglycosidase is heparanase Kit of item 10 or 11. 18. Kit useful for detecting heparan sulfate endoglycosidase in samples: A compartment is configured to receive one or more containers in a tightly packed condition. Carrier created; anti-heparan sulfate endoglycosidase a first container means containing the body; A detectably labeled protein that specifically binds to heparan sulfate endoglycosidase. A kit comprising a second container means containing the antibody. 19. Kit useful for detecting heparan sulfate endoglycosidase in samples: compartmented to receive one or more container means in a tightly packed condition; the carrier on which the painting was made; a first container containing an antibody that specifically binds to heparan sulfate endoglycosidase; means; a second containing detectably labeled heparan sulfate endoglycosidase; Kit containing container means. 20. said antibody in said first container means is immobilized on said container means; The kit according to claim 18 or 19. 21. If the antibody in the second container is radiolabeled, enzymatically labeled, fluorescently labeled, or 19. The kit of claim 18, labeled with a lomophore. 22. Claim wherein said antibody in said second container means is a monoclonal antibody. 18 kits. 23. Claims 18 to 18, wherein the heparan sulfate endoglycosidase is heparanase. Or 19 kits. 24. Claim 18 or 19, wherein the antibody in the first container means is polyclonal. kit. 25. Multiple containers with different amounts of heparan sulfate endoglycosidase antigen 20. The kit of claim 18 or 19, comprising a stage. 26. 20. A kit according to claim 18 or 19, wherein said container means is a tube. 27. A peptide of an antibody made immunologically against heparanase. The above peptide consists essentially of EEDLGKSREGSRTDD. 28. The peptide of claim 27, further comprising EEDLGKHSREGSRTDDC. The peptides defined above. 29. A method for producing antibodies that specifically bind to heparanase. A method comprising immunizing an animal with a carrier conjugated to a peptide from the terminal region . 30. 30. The method of claim 29, wherein the peptide is a peptide of claim 27 or 28. 31. 30. The method of claim 29, wherein the carrier is a protein. 32. 30. The method of claim 29, wherein the carrier is keyhole clam hemocyanin. .