EP0244932A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Solid-phase substrate yielding soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase
Assignee: The University of Texas System
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Filing: 1987-03-10
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Granted: —
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Expires: 2007-03-10
Abstract
Abstract A solid phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase and methods of producing said substrate are comprised in the present invention. The solid phase substrate comprises glycosaminoglycan bearing labeled substances bound to amino groups. The labeled glycosaminoglycan substrate is reductively aminated at its reducing terminal end to produce an amine-terminus. The substrate is further coupled to an amino-reactive solid matrix through its amine-terminus. A method of producing the solid phase substrate comprises the steps of: at least partially N-desulfating or N-deacylating a glycosaminoglycan; labeling at least partially N-deacylated or N-desulfated glycosaminoglycan with a substance yielding detectable signals to produce labeled glycosaminoglycan; completely N-acylating the labeled glycosaminoglycan with acyl anhydride or acyl halide; reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid phase support to produce the solid phase substrate. The solid phase substrate is usable to detect metastatic tumors by measurement of serum heparanase levels. The potential metastases of a tumor may also be determined by its heparanase levels.
Inventors
- 👤 Garth L. Nicolson
- 👤 Tatsuro Irimura (Nmi)
- 👤 Motowo Najima (Nmi)
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-04 12:27
CPC Classifications
C; C12; C12Y; C12Y302/00; C12Y302/01; C12Y302/01031; A; A61; A61K; A61K51/00; A61K51/12; C08; C08B; C08B37/00; C08B37/006; C08B37/0063; C08B37/0075; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12Q; C12Q1/00; C12Q1/34; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/573; G01N33/575; G01N33/5751; G01N33/5756; A61K2123/00; G01N2333/00; G01N2333/90; G01N2333/914; G01N2333/924; G01N2400/00; G01N2400/10; G01N2400/38; G01N2400/40
Claims Analysis
Total Claims: 99
Claim 1
Independent Claim
120 words
A method of producing a solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of glycosaminoglycan; (b) radiolabeling the at least partially N -desulfated or N -deacetylated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan; (c) completely N -acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled and N-acylated glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
Claim 2
Independent Claim
120 words
A method of producing a solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of glycosaminoglycan; (b) radiolabeling the at least partially N -desulfated or N -deacetylated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan; (c) completely N -acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled and N-acylated glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
Claim 3
Independent Claim
123 words
A method of producing a solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a heparan sulfate endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of heparan sulfate; (b) radiolabeling the at least partially N -desulfated or N -deacetylated heparan sulfate with radioisotopically labeled acetic anhydride to produce radioisotopically labeled heparan sulfate; (c) completely N-acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
Claim 4
Independent Claim
123 words
A method of producing a solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a heparan sulfate endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of heparan sulfate; (b) radiolabeling the at least partially N -desulfated or N -deacetylated heparan sulfate with radioisotopically labeled acetic anhydride to produce radioisotopically labeled heparan sulfate; (c) completely N-acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
Claim 5
Independent Claim
19 words
The method of claim l or 2 is characterized in that at least partially N-desulfating comprises solvolysis in dimethylsulfoxide.
Claim 6
Independent Claim
17 words
The method of claim l or 2 is characterized in that at least partially N-deacetylating comprises hydrazinolysis.
Claim 7
Independent Claim
24 words
The method of claim l or 2 is characterized in that the radioisotopically labeled acyl anhydride or acyl halide comprise ¹⁴C or ³H atoms.
Claim 8
Independent Claim
23 words
The method of claim l or 2 is characterized in that the radiolabeling step is carried out with ³H or ¹⁴C acetic anhydride.
Claim 9
Independent Claim
24 words
The method of claim l or 2 is characterized in that the reductive amination step comprises use of an ammonium salt and sodium cyanoborohydride.
Claim 10
Independent Claim
43 words
The method of claim l or 2 is characterized by an additional step is added of: (f) blocking uncoupled amine reactive sites on said solid phase support by aqueous incubation with an alkali metal borohydride or cyanoborohydride and a water soluble amine-bearing compound.
Claim 11
Independent Claim
37 words
The method of claim l or 2 is characterized by an additional step is added of: (f) blocking uncoupled amine-reactive sites on said solid-phase support by aqueous incubation with sodium cyanoborohydride and ethanolamine or glycine ethyl ester.
Claim 12
Dependent Claim
28 words
l0. The method of claim l characterized in that the glycosaminoglycan is hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or combinations thereof.
Claim 13
Dependent Claim
119 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of glycosaminoglycan; (b) radiolabeling the at least partially N -deacylated or N -desulfated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan; (c) completely N -acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 14
Dependent Claim
119 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of glycosaminoglycan; (b) radiolabeling the at least partially N -deacylated or N -desulfated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan; (c) completely N -acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said radioisotopically labeled glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 15
Dependent Claim
124 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -desulfating a quantity of heparan sulfate; (b) radiolabeling the at least partially N -desulfated heparan sulfate with radioisotopically labeled acetic anhydride or acetyl chloride to produce radioisotopically labeled heparan sulfate; (c) completely N -acylating the radioisotopically labelled heparan sulfate with an acyl anhydride or acyl halide. (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amine-reactive solid phase support to produce said solid phase substrate.
Claim 16
Dependent Claim
124 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -desulfating a quantity of heparan sulfate; (b) radiolabeling the at least partially N -desulfated heparan sulfate with radioisotopically labeled acetic anhydride or acetyl chloride to produce radioisotopically labeled heparan sulfate; (c) completely N -acylating the radioisotopically labelled heparan sulfate with an acyl anhydride or acyl halide. (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amine-reactive solid phase support to produce said solid phase substrate.
Claim 17
Dependent Claim
39 words
The solid-phase substrate of claim ll or l2 characterized in that the additional step is added of: (f) blocking any uncoupled amine-reactive sites on said solid-phase substrate by aqueous incubation with ethanolamine or glycine ethyl ester and sodium cyanoborohydride.
Claim 18
Dependent Claim
28 words
The solid-phase substrate of claim ll characterized in that the glycosaminoglycan is hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or combinations thereof.
Claim 19
Dependent Claim
50 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase said substrate characterized by glycosaminoglycan bearing radioisotopically labeled N-acyl groups, said glycosaminoglycan being reductively aminated at its reducing terminal end to produce an amine-terminus and being coupled to an amino-reactive solid matrix through said amine-terminus.
Claim 20
Dependent Claim
50 words
A solid-phase substrate which yields soluble radioisotopically labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase said substrate characterized by glycosaminoglycan bearing radioisotopically labeled N-acyl groups, said glycosaminoglycan being reductively aminated at its reducing terminal end to produce an amine-terminus and being coupled to an amino-reactive solid matrix through said amine-terminus.
Claim 21
Dependent Claim
16 words
The solid phase substrate of claim l5 characterized in that the acyl groups are acetyl groups.
Claim 22
Dependent Claim
18 words
The solid phase substrate of claim l5 characterized in that the labeled N-acyl groups comprise ³H or ¹⁴C.
Claim 23
Dependent Claim
20 words
The solid phase substrate of claim l5 characterized in that the glycosaminoglycan is heparan sulfate and the endoglycosidase is heparanase.
Claim 24
Dependent Claim
17 words
The solid phase substrate of claim l5 characterized in that the amine-reactive solid matrix comprises agarose beads.
Claim 25
Dependent Claim
20 words
The solid phase substrate of claim l5 characterized in that the amine-reactive solid matrix comprises cyanogen bromide activated agarose beads.
Claim 26
Dependent Claim
23 words
2l. The solid phase substrate of claim l5 characterized in that the amine-reactive solid matrix comprises an N -hydroxy succinide ester of agarose.
Claim 27
Dependent Claim
18 words
The solid phase substrate of claim l5 characterized in that the amine-reactive solid matrix comprises periodate activated agarose.
Claim 28
Dependent Claim
63 words
An assay procedure for measuring glycosaminoglycan endoglycosidase enzymic activity in a biological sample characterized by the steps of: (a) incubating a portion of the sample in a buffered aqueous mixture with radioactively labeled amine-terminal glycosaminoglycan bound solely through its terminal amino group to a solid phase; and (b) measuring radioactive label rendered soluble, this measurement being a function of glycosaminoglycan endoglycosidase enzymic activity.
Claim 29
Dependent Claim
63 words
An assay procedure for measuring glycosaminoglycan endoglycosidase enzymic activity in a biological sample characterized by the steps of: (a) incubating a portion of the sample in a buffered aqueous mixture with radioactively labeled amine-terminal glycosaminoglycan bound solely through its terminal amino group to a solid phase; and (b) measuring radioactive label rendered soluble, this measurement being a function of glycosaminoglycan endoglycosidase enzymic activity.
Claim 30
Dependent Claim
65 words
An assay procedure for measuring heparan sulfate endoglycosidase activity in a biological sample characterized by the steps of: (a) incubating a portion of the sample in a buffered aqueous mixture with radioactively labeled amino-terminal heparan sulfate bound solely through its terminal amine group to a solid phase; and (b) measuring radioactive labeled rendered soluble, this measurement being a function of heparan sulfate endoglycosidase enzymic activity.
Claim 31
Dependent Claim
65 words
An assay procedure for measuring heparan sulfate endoglycosidase activity in a biological sample characterized by the steps of: (a) incubating a portion of the sample in a buffered aqueous mixture with radioactively labeled amino-terminal heparan sulfate bound solely through its terminal amine group to a solid phase; and (b) measuring radioactive labeled rendered soluble, this measurement being a function of heparan sulfate endoglycosidase enzymic activity.
Claim 32
Independent Claim
110 words
A method of detecting the presence of metastatic tumor cells in a patient, the method characterized by: (a) obtaining serum samples from control individuals and from patients suspected of harboring metastatic tumor cells; (b) incubating portions of said serum samples in a buffered aqueous medium comprising an amine-terminal glycosaminoglycan labeled with radioisotopic N -acetyl groups, said labeled glycosaminoglycan being bound through its terminal amine to a matrix; (c) measuring levels of soluble radioisotopically labeled products formed as a function of incubation time; and (d) comparing levels of soluble radioisotopically labeled products formed by patient serum and by serum from control individuals, patients having metastatic tumor cells producing significantly higher levels.
Claim 33
Independent Claim
110 words
A method of detecting the presence of metastatic tumor cells in a patient, the method characterized by: (a) obtaining serum samples from control individuals and from patients suspected of harboring metastatic tumor cells; (b) incubating portions of said serum samples in a buffered aqueous medium comprising an amine-terminal glycosaminoglycan labeled with radioisotopic N -acetyl groups, said labeled glycosaminoglycan being bound through its terminal amine to a matrix; (c) measuring levels of soluble radioisotopically labeled products formed as a function of incubation time; and (d) comparing levels of soluble radioisotopically labeled products formed by patient serum and by serum from control individuals, patients having metastatic tumor cells producing significantly higher levels.
Claim 34
Dependent Claim
59 words
A solid phase substrate which yields soluble products labeled with a detectable signal upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate characterized by: a glycosaminoglycan bearing a label which does not prevent hydrolysis of the labeled glycosaminoglycan by a glycosaminoglycan endoglycosidase, said glycosaminoglycan being linked through its reducing terminal end by a single covalent linkage to a solid matrix.
Claim 35
Dependent Claim
59 words
A solid phase substrate which yields soluble products labeled with a detectable signal upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate characterized by: a glycosaminoglycan bearing a label which does not prevent hydrolysis of the labeled glycosaminoglycan by a glycosaminoglycan endoglycosidase, said glycosaminoglycan being linked through its reducing terminal end by a single covalent linkage to a solid matrix.
Claim 36
Independent Claim
119 words
A method of producing a solid-phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of glycosaminoglycan; (b) labeling amino groups of the at least partially N -desulfated or N -deacetylated glycosaminoglycan with a label yielding a detectable signal to product labeled glycosaminoglycan; (c) completely N -acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled and N-acylated glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
Claim 37
Independent Claim
119 words
A method of producing a solid-phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of glycosaminoglycan; (b) labeling amino groups of the at least partially N -desulfated or N -deacetylated glycosaminoglycan with a label yielding a detectable signal to product labeled glycosaminoglycan; (c) completely N -acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled and N-acylated glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
Claim 38
Independent Claim
125 words
A method of producing a solid-phase substrate which yields soluble labeled products upon hydrolysis by a heparan sulfate endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of heparan sulfate; (b) labeling amino groups of the at least partially N -desulfated or N -deacetylated heparan sulfate with a label yielding a detectable signal to produce labeled haparan sulfate; (c) completely N-acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal haparan sulfate; and (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
Claim 39
Independent Claim
125 words
A method of producing a solid-phase substrate which yields soluble labeled products upon hydrolysis by a heparan sulfate endoglycosidase, the method characterized by the steps of: (a) at least partially N -desulfating or N -deacetylating a quantity of heparan sulfate; (b) labeling amino groups of the at least partially N -desulfated or N -deacetylated heparan sulfate with a label yielding a detectable signal to produce labeled haparan sulfate; (c) completely N-acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal haparan sulfate; and (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
Claim 40
Independent Claim
27 words
The method of claims 27 or 28 characterized in that the labeling step is carried out with a fluorescent label, an enzyme label or a radioisotopic label.
Claim 41
Dependent Claim
118 words
A solid-phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of glycosaminoglycan; (b) labeling amino groups of the at least partially N -deacylated or N -desulfated glycosaminoglycan with a substance yielding a detectable signal to produce labeled glycosaminoglycan; (c) completely N -acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 42
Dependent Claim
118 words
A solid-phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of glycosaminoglycan; (b) labeling amino groups of the at least partially N -deacylated or N -desulfated glycosaminoglycan with a substance yielding a detectable signal to produce labeled glycosaminoglycan; (c) completely N -acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 43
Dependent Claim
128 words
3l. A solid-phase substrate which yields soluble labeled products upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of heparan sulfate; (b) labeling amino groups of the at least partially N -deacetylated or N -desulfated heparan sulfate with a substance yielding a detectable signal to produce labeled heparan sulfate; (c) completely N -acylating amino groups of the labeled heparan sulfate with an acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 44
Dependent Claim
128 words
3l. A solid-phase substrate which yields soluble labeled products upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process characterized by the steps of: (a) at least partially N -deacylating or N -desulfating a quantity of heparan sulfate; (b) labeling amino groups of the at least partially N -deacetylated or N -desulfated heparan sulfate with a substance yielding a detectable signal to produce labeled heparan sulfate; (c) completely N -acylating amino groups of the labeled heparan sulfate with an acyl anhydride or acyl halide; (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal heparan sulfate; and (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid-phase support to produce said solid-phase substrate.
Claim 45
Dependent Claim
25 words
The solid phase substrate of claim 30 or 3l characterized in that the label is a fluorescent label, a radioisotopic label or an enzymatic label.
Claim 46
Dependent Claim
46 words
A solid phase substrate which, upon hydrolysis by a glycosaminoglycan endoglycosidase, yields soluble products with a detectable label, the substrate characterized by a glycosaminoglycan, through amino groups thereof, bearing a detectable label, said glycosaminoglycan being bound through its aminated reducing terminal hexose to a solid matrix.
Claim 47
Dependent Claim
46 words
A solid phase substrate which, upon hydrolysis by a glycosaminoglycan endoglycosidase, yields soluble products with a detectable label, the substrate characterized by a glycosaminoglycan, through amino groups thereof, bearing a detectable label, said glycosaminoglycan being bound through its aminated reducing terminal hexose to a solid matrix.
Claim 48
Independent Claim
140 words
A method of producing a liquid-phase substrate which, upon hydrolysis by a glycosaminoglycan endoglycosidase, yields labeled products, and using said substrate to assay human glycosaminoglycan endoglycosidase, the method is characterized by the steps of: (a) labeling a glycosaminoglycan at one or more sites with a label yielding a detectable signal; (b) tagging the labeled glycosaminoglycan with a molecule at a site on the glycosaminoglycan that has not been labeled; (c) incubating the labeled and tagged glycosaminoglycan in a buffered aqueous solution with a human biological sample suspected of containing glycosaminoglycan endoglycosidase; (d) separating any labeled untagged products resulting from glycosaminoglycan endoglycosidase-induced hydrolysis of the labeled and tagged glycosaminoglycan substrate from tagged products and unaltered labeled and tagged glycosaminoglycan substrate; and (e) determining amounts of labeled untagged product, said amounts being proportional to glycosaminoglycan endoglycosidase levels in the human biological sample.
Claim 49
Independent Claim
140 words
A method of producing a liquid-phase substrate which, upon hydrolysis by a glycosaminoglycan endoglycosidase, yields labeled products, and using said substrate to assay human glycosaminoglycan endoglycosidase, the method is characterized by the steps of: (a) labeling a glycosaminoglycan at one or more sites with a label yielding a detectable signal; (b) tagging the labeled glycosaminoglycan with a molecule at a site on the glycosaminoglycan that has not been labeled; (c) incubating the labeled and tagged glycosaminoglycan in a buffered aqueous solution with a human biological sample suspected of containing glycosaminoglycan endoglycosidase; (d) separating any labeled untagged products resulting from glycosaminoglycan endoglycosidase-induced hydrolysis of the labeled and tagged glycosaminoglycan substrate from tagged products and unaltered labeled and tagged glycosaminoglycan substrate; and (e) determining amounts of labeled untagged product, said amounts being proportional to glycosaminoglycan endoglycosidase levels in the human biological sample.
Claim 50
Dependent Claim
33 words
The method of claim 34 characterized in that the separating step involves use of an antibody raised to the tagging molecule or use of a binding protein which binds to the tagging molecule.
Claim 51
Dependent Claim
19 words
The method of claim 35 characterized in that the antibody or binding protein is coupled to a solid-phase support.
Claim 52
Dependent Claim
60 words
An immunoassay method for detecting the presence of a glycosaminoglycan endoglycosidase characterized by combining a buffered aqueous solution of a human biological sample suspected of containing glycosaminoglycan endoglycosidase with an antibody or antibodies raised to the glycosaminoglycan endoglycosidase and determining the level of binding of said antibody or antibodies to the sample as indicative of the presence of glycosaminoglycan endoglycosidase.
Claim 53
Dependent Claim
60 words
An immunoassay method for detecting the presence of a glycosaminoglycan endoglycosidase characterized by combining a buffered aqueous solution of a human biological sample suspected of containing glycosaminoglycan endoglycosidase with an antibody or antibodies raised to the glycosaminoglycan endoglycosidase and determining the level of binding of said antibody or antibodies to the sample as indicative of the presence of glycosaminoglycan endoglycosidase.
Claim 54
Dependent Claim
14 words
The method of claim 37 characterized in that the antibody or antibodies are monoclonal.
Claim 55
Dependent Claim
20 words
The method of claim 37 characterized in that the immunoassay is a radioimmunoassay, an enzyme immunoassay, or a fluorescent immunoassay.
Claim 56
Independent Claim
136 words
A method of producing a liquid-phase substrate which, upon hydrolysis by a heparan sulfate endoglycosidase, yields labeled products and using said substrate to assay human heparan sulfate endoglycosidase, the method is characterized by the steps of: (a) labeling heparan sulfate at one or more sites with a label yielding a detectable signal; (b) tagging the labeled heparan sulfate with a molecule at a site on the heparan sulfate that has not been labeled; (c) incubating the labeled and tagged heparan sulfate in a buffered aqueous solution with a human biological sample suspected of containing heparan sulfate endoglycosidase; (d) separating any labeled untagged products resulting from heparan-sulfate endoglycosidaseinduced of the labeled and tagged heparan sulfate; and (e) determining amounts of labeled untagged product, said amounts being proportional to heparan sulfate endoglycosidase levels in the human biological sample.
Claim 57
Independent Claim
136 words
A method of producing a liquid-phase substrate which, upon hydrolysis by a heparan sulfate endoglycosidase, yields labeled products and using said substrate to assay human heparan sulfate endoglycosidase, the method is characterized by the steps of: (a) labeling heparan sulfate at one or more sites with a label yielding a detectable signal; (b) tagging the labeled heparan sulfate with a molecule at a site on the heparan sulfate that has not been labeled; (c) incubating the labeled and tagged heparan sulfate in a buffered aqueous solution with a human biological sample suspected of containing heparan sulfate endoglycosidase; (d) separating any labeled untagged products resulting from heparan-sulfate endoglycosidaseinduced of the labeled and tagged heparan sulfate; and (e) determining amounts of labeled untagged product, said amounts being proportional to heparan sulfate endoglycosidase levels in the human biological sample.
Claim 58
Dependent Claim
38 words
4l. The method of claim 40 characterized in that the separation is achieved by an antibody raised to the molecule used as a tag or by a binding protein which binds to the molecule used as a tag.
Claim 59
Independent Claim
19 words
The method of claim 4l characterized in that the antibody or binding protein is coupled to a solid-phase support.
Claim 60
Dependent Claim
64 words
An immunoassay method for detecting the presence of a heparan sulfate endoglycosidase characterized by combining a buffered aqueous solution of a human biological sample suspected of containing heparan sulfate endoglycosidase with an antibody or antibodies raised to the heparan sulfate endoglycosidase and determining the level of binding of said antibody or antibodies to the sample as indicative of the presence of heparan sulfate endoglycosidase.
Claim 61
Dependent Claim
64 words
An immunoassay method for detecting the presence of a heparan sulfate endoglycosidase characterized by combining a buffered aqueous solution of a human biological sample suspected of containing heparan sulfate endoglycosidase with an antibody or antibodies raised to the heparan sulfate endoglycosidase and determining the level of binding of said antibody or antibodies to the sample as indicative of the presence of heparan sulfate endoglycosidase.
Claim 62
Dependent Claim
15 words
The method of claim 43 characterized in that the antibody or antibodies are monoclonal antibodies.
Claim 63
Dependent Claim
20 words
The method of claim 43 characterized in that the immunoassay is a radioimmunoassay, an enzyme immunoassay, or a fluorescent immunoassay.
Claim 64
Dependent Claim
62 words
A kit useful for the detection of a glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the glycosaminoglycan endoglycosidase; a second container means comprising a detectably labeled antibody which is specific for the glycosaminoglycan endoglycosidase.
Claim 65
Dependent Claim
62 words
A kit useful for the detection of a glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the glycosaminoglycan endoglycosidase; a second container means comprising a detectably labeled antibody which is specific for the glycosaminoglycan endoglycosidase.
Claim 66
Dependent Claim
54 words
A kit useful for the detection of glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the glycosaminoglycan endoglycosidase; a second container means comprising detectably labeled glycosaminoglycan endoglycosidase.
Claim 67
Dependent Claim
54 words
A kit useful for the detection of glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the glycosaminoglycan endoglycosidase; a second container means comprising detectably labeled glycosaminoglycan endoglycosidase.
Claim 68
Dependent Claim
25 words
The kit of any of claims 46 or 47 characterized in that said antibody in said first container means is immobilized on said container means.
Claim 69
Dependent Claim
28 words
The kit of claim 46 characterized in that said antibody in said second container is labeled with a radiolabel, an enzyme label, a fluorescent label or a chromophore.
Claim 70
Dependent Claim
23 words
The kit of claim 46 characterized in that the antibody in said first container means or second container means is a monoclonal antibody.
Claim 71
Dependent Claim
25 words
5l. The kit of claims 46 or 47 which also is characterized by a multiplicity of container means with different amounts of glycosaminoglycan endoglycosidase antigen.
Claim 72
Dependent Claim
23 words
The kit of any of claims 46 or 47 characterized in that said first container means or second container means is a tube.
Claim 73
Dependent Claim
65 words
A kit useful for the detection of a heparan sulfate endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the heparan sulfate endoglycosidase; a second container means comprising a detectably labeled antibody which is specific for the heparan sulfate endoglycosidase.
Claim 74
Dependent Claim
65 words
A kit useful for the detection of a heparan sulfate endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising an antibody which is specific for the heparan sulfate endoglycosidase; a second container means comprising a detectably labeled antibody which is specific for the heparan sulfate endoglycosidase.
Claim 75
Dependent Claim
60 words
A kit useful for the detection of heparan sulfate endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means characterized by an antibody which is specific for the heparan sulfate endoglycosidase; a second container means characterized by detectably labeled heparan sulfate endoglycosidase antigen.
Claim 76
Dependent Claim
60 words
A kit useful for the detection of heparan sulfate endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means characterized by an antibody which is specific for the heparan sulfate endoglycosidase; a second container means characterized by detectably labeled heparan sulfate endoglycosidase antigen.
Claim 77
Dependent Claim
24 words
The kit of any of claims 53 or 54 characterized by said antibody in said first container means is immobilized on said container means.
Claim 78
Dependent Claim
27 words
The kit of claim 53 characterized by said antibody in said second container is labeled with a radiolabel, an enzyme label, a fluorescent label or a chromophore.
Claim 79
Dependent Claim
18 words
The kit of claim 53 characterized by said antibody in said second container means is a monoclonal antibody.
Claim 80
Dependent Claim
25 words
The kit of claims 53 or 54 which also is characterized by a multiplicity of container means with different amounts of heparan sulfate endoglycosidase antigen.
Claim 81
Dependent Claim
18 words
The kit of any of claims 53 or 54 characterized in that said container means is a tube.
Claim 82
Independent Claim
159 words
A method for producing a soluble glycosaminoglycan substrate comprising a label and a tagging molecule, the glycosamin glycan substrate, upon hydrolysis by a glycosaminoglycan endoglycosidase, yielding soluble products comprising a label and soluble products comprising a tagging molecule, the soluble products comprising a tagging molecule and unhydrolyzed glycosaminoglycan substrate being extractable from solution upon exposure to a solid phase protein having binding affinity for the tagging molecule, the method is characterized by the steps of: (a) labeling a glycosaminoglycan substrate for glycosaminoglycan endoglycosidase at one or more sites with a label which yields a detectable signal, said label not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate; (b) binding the labeled glycosaminoglycan substrate to a tagging molecule at a site on the glycosaminoglycan substrate that has not been labeled, the tagging molecule having affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding glycosaminoglycan endoglycosidase- induced hydrolysis of glycosaminoglycan substrate.
Claim 83
Independent Claim
159 words
A method for producing a soluble glycosaminoglycan substrate comprising a label and a tagging molecule, the glycosamin glycan substrate, upon hydrolysis by a glycosaminoglycan endoglycosidase, yielding soluble products comprising a label and soluble products comprising a tagging molecule, the soluble products comprising a tagging molecule and unhydrolyzed glycosaminoglycan substrate being extractable from solution upon exposure to a solid phase protein having binding affinity for the tagging molecule, the method is characterized by the steps of: (a) labeling a glycosaminoglycan substrate for glycosaminoglycan endoglycosidase at one or more sites with a label which yields a detectable signal, said label not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate; (b) binding the labeled glycosaminoglycan substrate to a tagging molecule at a site on the glycosaminoglycan substrate that has not been labeled, the tagging molecule having affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding glycosaminoglycan endoglycosidase- induced hydrolysis of glycosaminoglycan substrate.
Claim 84
Dependent Claim
163 words
6l. A method for assaying a glycosaminoglycan endoglycosidase characterized by the steps of: (a) labeling a glycosaminoglycan substrate for glycosaminoglycan endoglycosidase at one or more sites with a label which yields a detectable signal, said label not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate; (b) binding the labeled glycosaminoglycan substrate to a tagging molecule at a site on the labeled glycosaminoglycan substrate that has not been labeled, the tagging molecule having affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the labeled and tagged glycosaminoglycan substrate; (c) incubating the labeled and tagged glycosaminoglycan substrate with a buffered aqueous solution comprising a human biological sample suspected of containing glycosaminoglycan endoglycosidase; and (d) separating glycosaminoglycan substrate and tagged products resulting from glycosaminoglycan endoglycosidase-induced hydrolysis from untagged labeled products resulting from the hydrolysis, said separation involving binding of glycosaminoglycan-bound tagging molecules to a protein having an affinity for the tagging molecule.
Claim 85
Dependent Claim
163 words
6l. A method for assaying a glycosaminoglycan endoglycosidase characterized by the steps of: (a) labeling a glycosaminoglycan substrate for glycosaminoglycan endoglycosidase at one or more sites with a label which yields a detectable signal, said label not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the glycosaminoglycan substrate; (b) binding the labeled glycosaminoglycan substrate to a tagging molecule at a site on the labeled glycosaminoglycan substrate that has not been labeled, the tagging molecule having affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding glycosaminoglycan endoglycosidase-induced hydrolysis of the labeled and tagged glycosaminoglycan substrate; (c) incubating the labeled and tagged glycosaminoglycan substrate with a buffered aqueous solution comprising a human biological sample suspected of containing glycosaminoglycan endoglycosidase; and (d) separating glycosaminoglycan substrate and tagged products resulting from glycosaminoglycan endoglycosidase-induced hydrolysis from untagged labeled products resulting from the hydrolysis, said separation involving binding of glycosaminoglycan-bound tagging molecules to a protein having an affinity for the tagging molecule.
Claim 86
Independent Claim
156 words
A method for producing a soluble heparan sulfate derivative comprising a label and a tagging molecule, the heparan sulfate derivative, upon hydrolysis by a heparanase, yielding soluble products comprising a label and soluble products comprising a tagging molecule, the soluble products comprising a tagging molecule being extractable from solution upon exposure to a solid phase protein having binding affinity for the tagging molecule, the method characterized by the steps of: (a) labeling a heparan sulfate substrate for heparanase at one or more sites with a label which yields a detectable signal said label not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (b) binding the labeled heparan sulfate substrate to a tagging molecule at a site on the heparan sulfate that has not been labeled, the tagging molecule having binding affinity for a protein which may be attached to a solid matrix, aaid tagging molecule not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate.
Claim 87
Independent Claim
156 words
A method for producing a soluble heparan sulfate derivative comprising a label and a tagging molecule, the heparan sulfate derivative, upon hydrolysis by a heparanase, yielding soluble products comprising a label and soluble products comprising a tagging molecule, the soluble products comprising a tagging molecule being extractable from solution upon exposure to a solid phase protein having binding affinity for the tagging molecule, the method characterized by the steps of: (a) labeling a heparan sulfate substrate for heparanase at one or more sites with a label which yields a detectable signal said label not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (b) binding the labeled heparan sulfate substrate to a tagging molecule at a site on the heparan sulfate that has not been labeled, the tagging molecule having binding affinity for a protein which may be attached to a solid matrix, aaid tagging molecule not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate.
Claim 88
Independent Claim
159 words
A method for assaying a heparanase characterized by the steps of: (a) labeling a heparan sulfate substrate for heparanase at one or more sites with a label which yields a detectable signal said label not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (b) tagging the labeled heparan sulfate substrate with a tagging molecule at a site on the heparan sulfate that has not been labeled, the tagging molecule having binding affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (c) incubating the labeled and tagged heparan sulfate substrate with a buffered aqueous solution comprising a human biological sample suspected of containing heparanase; and (d) separating heparan sulfate substrate and tagged products resulting from heparanase-induced hydrolysis from untagged labeled products resulting from the hydrolysis, said separation involving binding of tagged molecules to a protein having a binding affinity for the tagging molecule.
Claim 89
Independent Claim
159 words
A method for assaying a heparanase characterized by the steps of: (a) labeling a heparan sulfate substrate for heparanase at one or more sites with a label which yields a detectable signal said label not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (b) tagging the labeled heparan sulfate substrate with a tagging molecule at a site on the heparan sulfate that has not been labeled, the tagging molecule having binding affinity for a protein which may be attached to a solid matrix, said tagging molecule not substantially impeding heparanase-induced hydrolysis of the heparan sulfate substrate; (c) incubating the labeled and tagged heparan sulfate substrate with a buffered aqueous solution comprising a human biological sample suspected of containing heparanase; and (d) separating heparan sulfate substrate and tagged products resulting from heparanase-induced hydrolysis from untagged labeled products resulting from the hydrolysis, said separation involving binding of tagged molecules to a protein having a binding affinity for the tagging molecule.
Claim 90
Independent Claim
26 words
The method of claim 6l or 63 characterized by the protein having a binding affinity for the bound tagging molecule is affixed to a solid matrix.
Claim 91
Dependent Claim
69 words
A kit useful for the detection of glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising substrate for glycosaminoglycan endoglycosidase, said substrate bearing a tagging molecule and a detectable label; a second container means comprising a protein which has specific binding affinity for the tagging molecule of the substrate.
Claim 92
Dependent Claim
69 words
A kit useful for the detection of glycosaminoglycan endoglycosidase in a sample which is characterized by: a carrier being compartmentalized to receive one or more container means in close confinement therein; a first container means comprising substrate for glycosaminoglycan endoglycosidase, said substrate bearing a tagging molecule and a detectable label; a second container means comprising a protein which has specific binding affinity for the tagging molecule of the substrate.
Claim 93
Dependent Claim
14 words
The kit of claim 65 characterized in that said protein is a monoclonal antibody.
Claim 94
Dependent Claim
22 words
The kit of claim 66 characterized in that said monoclonal antibody in said first container means is immobilized on said container means.
Claim 95
Dependent Claim
23 words
The kit of claim 65 characterized in that said detectable label is a radiolabel, an enzyme label, a fluorescent label or a chromophore.
Claim 96
Dependent Claim
13 words
The kit of claim 65 characterized in that said protein is an antibody.
Claim 97
Dependent Claim
22 words
The kit of claim 65 which also is characterized by a multiplicity of container means with different amounts of glycosaminoglycan endoglycosidase antigen.
Claim 98
Dependent Claim
22 words
7l. The kit of claim 65 which also is characterized by a multiplicity of container means with different amounts of detectable label.
Claim 99
Dependent Claim
13 words
The kit of claim 65 characterized in that said container means are tubes.
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