Assays for detection of glycosaminoglycans
View Patent ↗Disclosed herein are novel methods, assays and kits useful for the diagnosis and monitoring of subjects with mucopolysaccharidoses (MPS), The methods, assays and kits are particularly useful for detecting the presence of one or more glycosaminoglycans which correlate to MPS and its severity in a variety of biological samples.
1. A method for determining the concentration of one or both of dermatan sulfate (DS) and heparin sulfate (HS) in a sample, the method comprising (a) treating a sample with at least one of chondroitinase B and one or more heparineses, (b) combining a serine protease shown in FIG. 7 or 8 a labeled substrate for said serine protease, an inhibitor of said serine protease, and the sample under conditions and for a time suitable for cleavage of the labeled substrate by the serine protease to produce a detectable signal, (c) detecting the detectable signal, and (d) comparing the amount of detectable signal with a standard to determine the concentration of said one or both of DS and HS in said sample.
2. The method according to claim 1 , wherein the labeled substrate is a chromogenic or fluorogenic substrate.
3. The method according to claim 1 , wherein the serine protease is thrombin and the labeled substrate is a chromogenic thrombin substrate.
4. The method according to claim 1 , wherein detecting the detectable signal is performed by spectrophotometric detection.
5. The method according to claim 1 , wherein the standard is a curve which calibrates spectrophotometric absorbance with glycosaminoglycan concentration.
6. The method of claim 1 , wherein said inhibitor of said serine protease is selected from the group consisting of heparin cofactor II and antithrombin III.
7. A method for determining the concentration of one or both of dermatan sulfate (DS) and heparan sulfate (HS) in a sample, the method comprising (a) combining a serine protease shown in FIG. 7 or FIG. 8 , a labeled substrate for said serine protease, an inhibitor of said serine protease, and a cerebrospinal fluid sample under conditions and for a time suitable for cleavage of the labeled substrate by the serine protease to produce a detectable signal, (b) detecting the detectable signal, and (c) comparing the amount of detectable signal with a standard to determine the concentration of said one or both of DS and HS in said sample.
8. The method of claim 7 , wherein the labeled substrate is a chromogenic or fluorogenic substrate.
9. The method of claim 7 , wherein the serine protease is thrombin and the labeled substrate is a chromogenic thrombin substrate.
10. The method of claim 7 , wherein detected the detectable signal is performed by spectrophotometric detection.
11. The method of claim 7 , wherein the standard is a curve which calibrates spectrophotometric absorbance with glycosaminoglycan concentration.
12. The method of claim 7 , wherein said inhibitor of said serine protease is selected from the group consisting of heparin cofactor II and antithrombin III.
13. The method of claim 7 , wherein the sample is treated with at least one of chondroitinase B and one or more heparinases.