Methods relating to testing for lysosomal storage disorders
View Patent ↗Processes are provided that include additive compounds suitable for detecting the activity of an enzyme such as a lysosomal storage enzyme where the additive is a salt of oleic acid. Inclusion of a salt of oleic acid unexpectedly improves enzyme activity and reproducibility.
1. A process for detecting enzymatic activity in vitro, comprising:
contacting a sample containing a target enzyme with a substrate and one or more oleate salts wherein said salt is selected from the group consisting of sodium, magnesium, potassium, calcium, lithium and zinc, said step of contacting is performed under conditions wherein the target enzyme is lysosomal storage enzyme capable of acting on the substrate to produce an enzymatic product, and wherein said oleate salt allows for improved enzyme activity, detection of lower enzyme amounts, and/or greater reproducibility; and
detecting said enzymatic product, wherein said target enzyme is acid α-galactosidase A, acid β-glucocerebrosidase, galactocerebrosidase, acid α-glucosidase, acid sphingomylenase, α-L-iduronidase, or combinations thereof.
2. The process of claim 1 wherein said oleate salt comprises a monovalent salt.
3. The process of claim 1 wherein said oleate salt comprises a divalent salt.
4. The process of claim 3 wherein said divalent salt is magnesium, calcium, or zinc.
5. The process of claim 1 wherein said oleate salt is selected from the group consisting of sodium oleate, potassium oleate, magnesium oleate, calcium oleate, or lithium oleate.
6. The process of claim 1 wherein said step of detecting is by mass spectrometry.
7. The process of claim 1 further comprising contacting said sample with an internal standard.
8. The process of claim 7 wherein said internal standard comprises deuterium.
9. The process of claim 1 further comprising diagnosing a subject with a lysosomal storage disorder.
10. The process of claim 1 , wherein the process excludes the addition of oleic acid.
11. A process for detecting enzymatic activity in vitro, comprising:
preparing a substrate component comprising combining a substrate and an oleate salt wherein said salt is selected from the group consisting of sodium, magnesium, potassium, calcium, lithium and zinc;
drying said substrate component;
contacting a sample containing a target enzyme with said substrate component in an aqueous buffer, said step of contacting is performed under conditions wherein the target enzyme is lysosomal storage enzyme capable of acting on the substrate to produce an enzymatic product; and wherein said oleate salt allows for improved enzyme activity, detection of lower enzyme amounts, and/or greater reproducibility; and
detecting said enzymatic product, wherein said target enzyme is acid α-galactosidase A, acid β-glucocerebrosidase, galactocerebrosidase, acid α-glucosidase, acid sphingomylenase, α-L-iduronidase, or combinations thereof.
12. The process of claim 11 wherein said oleate salt is selected from the group consisting of sodium oleate, potassium oleate, magnesium oleate, calcium oleate, or lithium oleate.
13. The process of claim 11 wherein said aqueous buffer comprises succinic acid.
14. The process of claim 11 wherein said aqueous buffer has a pH of 4.3 to 4.0.
15. The process of claim 11 wherein said step of contacting further comprises adding an internal standard to said substrate and said oleate salt prior to drying.
16. The process of claim 11 further comprising combining said substrate component and said aqueous buffer prior to said step of contacting.
17. The process of claim 11 wherein said step of detecting is by mass spectrometry.
18. The process of claim 11 further comprising diagnosing a subject from which said sample is derived with the presence or absence of a lysosomal storage disorder.
19. The process of claim 11 , wherein the process excludes the addition of oleic acid.