Methods and compositions for assaying the activity of one or more lysosomal enzymes

Patent metadata

Field	Value
Publication number	US-9512463-B2
Application number	US-201214352291-A
Country	US
Kind code	B2
Filing date	Nov 8, 2012
Priority date	Nov 8, 2011
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

Methods and liquid compositions for assaying the activity of one or more lysosomal enzymes in a sample are provided. In some embodiments, the assay is a multiplexed assay for the activities of a plurality of lysosomal enzymes in the sample. The compositions and methods can comprise or employ: one or more metal cations effective for precipitating sulfate ions, one or more metal cations effective for precipitating phosphate ions, a maltase glucoamylase inhibitor, a beta-N-acetylhexosaminidase inhibitor, and one or more surfactants.

First claim

The invention claimed is:

1. A method for assaying enzymatic activity of one or more lysosomal enzymes, comprising:
(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes;
(b) adding an enzyme substrate for each lysosomal enzyme to be analyzed to the solution comprising the enzymes and incubating the substrates with the enzymes in an enzyme reaction solution for a time sufficient to provide a solution comprising an enzyme product for each lysosomal enzyme present in the sample, wherein the enzyme reaction solution comprises:
(i) one or more metal cations effective for precipitating sulfate ions;
(ii) one or more metal cations effective for precipitating phosphate ions;
(iii) a maltase glucoamylase inhibitor;
(iv) a beta-N-acetylhexosaminidase inhibitor; and
(v) one or more surfactants;

(c) optionally quenching the enzyme reaction;
(d) determining the quantities of the enzyme products; and
(e) determining the enzymatic activity of the one or more lysosomal enzymes based on the quantities determined in step (d).

2. The method of claim 1 , further comprising adding an internal standard for each lysosomal enzyme to be analyzed before, after, or simultaneously with the addition of substrates.

3. The method of claim 2 , wherein determining the quantities of the enzyme products comprises determining the ratio of each product to its internal standard by mass spectrometric analysis.

4. The method of claim 3 , wherein the mass spectrometric analysis is tandem mass spectrometric analysis.

5. The method of claim 3 , wherein determining the quantities of the products comprises tandem mass spectrometric analysis in which the parent ions of the products and their internal standards are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions.

6. The method of claim 5 , wherein determining the quantities of the products comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the products.

7. The method of claim 3 , wherein determining the quantities of the enzyme products comprises conducting the solution comprising the enzyme product to a mass spectrometer by liquid chromatography or flow injection.

8. The method of claim 1 , wherein the sample is a blood sample, a tissue sample, or a dried blood spot.

9. The method of claim 8 , further comprising using the quantities of the products to determine whether the dried blood sample is from a candidate for treatment for a condition associated with one or more lysosomal enzyme deficiencies.

10. The method of claim 1 , wherein the one or more lysosomal enzymes comprise an enzyme selected from the group consisting of:
(a) α-glucosidase (GAA);
(b) α-galactosidase (GLA);
(c) α-L-iduronidase (IDUA);
(d) β-glucocerebrosidase (ABG);
(e) β-galactocerebrosidase (GALC);
(f) sphingomyelinase (ASM);
(g) iduronate 2-sufatase (ID2S);
(h) N-acetylgalactosamine 6-sulfatase (GALES); and
(i) N-acetylgalactosamine 4-sulfatase (GAL4S).

11. The method of claim 1 , wherein the enzyme reaction solution further comprises a buffer.

12. An aqueous composition, comprising:
(a) one or more metal cations effective for precipitating sulfate ions;
(b) one or more metal cations effective for precipitating phosphate ions;
(c) a maltase glucoamylase inhibitor;
(d) a beta-N-acetylhexosaminidase inhibitor; and
(e) one or more surfactants.

13. The composition of claim 12 , further comprising a buffer.

14. The composition of claim 12 , wherein the metal cation effective for binding sulfate ions is selected from the group consisting of Ba2+, Ce3+, Hg+, Pb2+, Ra2+, Sr2+, Bi3+, Cd2+, Ca2+, and Mg2+.

15. The composition of claim 12 , wherein the metal cation effective for binding phosphate ions is Ba2+, Ce3+, Hg+, Pb2+, Ra2+, Sr2+, Bi3+, Cd2+, Ca2+, and Mg2+.

16. The composition of claim 12 , wherein the maltase glucoamylase inhibitor is acarbose.

17. The composition of claim 12 , wherein the beta-N-acetylhexosaminidase inhibitor is 2-acetamido-2-deoxy-D-glucono-1,5-lactone.

18. The composition of claim 12 , having a pH from about 2 to about 9.

19. The composition of claim 12 , further comprising one or more substrates for a lysosomal enzyme selected from the group consisting of:
(a) α-glucosidase (GAA);
(b) α-galactosidase (GLA);
(c) α-L-iduronidase (IDUA);
(d) β-glucocerebrosidase (ABG);
(e) β-galactocerebrosidase (GALC);
(f) sphingomyelinase (ASM);
(g) iduronate 2-sufatase (ID2S);
(h) N-acetylgalactosamine 6-sulfatase (GALES); and
(i) N-acetylgalactosamine 4-sulfatase (GAL4S).

20. The composition of claim 19 , further comprising one or more internal standards for a lysosomal enzyme selected from the group consisting of:
(a) α-glucosidase (GAA);
(b) α-galactosidase (GLA);
(c) α-L-iduronidase (IDUA);
(d) β-glucocerebrosidase (ABG);
(e) β-galactocerebrosidase (GALC);
(f) sphingomyelinase (ASM);
(g) iduronate 2-sufatase (ID2S);
(h) N-acetylgalactosamine 6-sulfatase (GALES); and
(i) N-acetylgalactosamine 4-sulfatase (GAL4S).

Assignees

• Univ Washington Through Its Center For Commercialization
• Univ Washington

Inventors

• Gelb Michael H
• Turecek Frantisek
• Spacil Zdenek
• Scott C Ronald
• Barcenas Rodriguez Mariana Natali

Classifications

• G01N30/7233

  interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04) · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C12Q2545/114

  involving a quantitation step · CPC title

• G01N33/573

  for enzymes or isoenzymes · CPC title

• C12Q1/40 Primary

  involving amylase · CPC title