Assay for B-galactosidase activity

• US 9,012,165 B2
• Filed: 11/25/2013
• Issued: 04/21/2015
• Est. Priority Date: 03/22/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Eckhardt, Allen E.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US14/089,070
• Publication Number:
  US 20140141460A1
• Time in Patent Office: 512 Days
• Field of Search: 435/14, 347/68
• US Class Current: 435/14
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 311/18 substituted otherwise than in position 3 or 7
• CPC Class Codes: C07D 311/20 hydrogenated in the hetero ring
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 413/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C07H 17/075 Benzo[b]pyran-2-ones
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/40 involving amylase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2333/916 : acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2333/926 : acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
• CPC Class Codes: G01N 2333/938 : acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
• CPC Class Codes: G01N 2800/04 : Endocrine or metabolic disorders
• CPC Class Codes: G01N 33/5438 : Electrodes

Abstract

The invention is directed to an assay for acid β-galactosidase activity. The invention may include combining in oil a sample droplet with a 4-methylumbelliferyl-B-galactose to yield a reaction droplet; splitting the reaction droplet to yield a first daughter droplet and a second daughter droplet; combining the first daughter droplet with a stop buffer droplet to yield a first stopped reaction droplet; incubating the second daughter droplet; combining the second daughter droplet with a stop buffer droplet to yield a second stopped reaction droplet; and measuring 4-methylumbelliferone released in the first and second stopped reaction droplets.

Claims

1. An assay for acid β

-galactosidase activity, comprising;

(a) combining in oil a sample droplet with a 4-methylumbelliferyl-B-galactose to yield a reaction droplet;

(b) splitting the reaction droplet to yield a first daughter droplet and a second daughter droplet;

(c) combining the first daughter droplet with a stop buffer droplet to yield a first stopped reaction droplet;

(d) incubating the second daughter droplet;

(e) combining the second daughter droplet with a stop buffer droplet to yield a second stopped reaction droplet; and

(f) measuring 4-methylumbelliferone released in the first and second stopped reaction droplets;

(g) correlating the 4-methylumbelliferone measured in the first and second stopped reaction droplets with the acid beta-galactosidase activity.

2. The method of claim 1 wherein multiple assays for β

-galactosidase activity are performed in parallel.

3. The method of claim 1 wherein the incubating proceeds for a time which is less than about 24 hours.

4. The method of claim 1 wherein the incubating proceeds for a time which is less than about 12 hours.

5. The method of claim 1 wherein the incubating proceeds for a time which is less than about 9 hours.

6. The method of claim 1 wherein the incubating proceeds for a time which is less than about 6 hours.

7. The method of claim 1 wherein the incubating proceeds for a time which is less than about 3 hours.

8. The method of claim 1 wherein the incubating proceeds at about room temperature.

9. The method of claim 1 wherein the droplets are surrounded by oil.

10. The method of claim 1 wherein the steps of the method are performed in droplets controlled by a droplet actuator.

11. The method of claim 10 wherein the droplet actuator controls the steps using electrode mediated droplet operations.

12. The method of claim 11 wherein the droplet actuator controls the steps using electrowetting mediated droplet operations.

13. The method of claim 11 wherein the droplet actuator controls the steps using dielectrophoresis mediated droplet operations.

14. The method of claim 1 wherein the sample comprises a blood sample.

15. The method of claim 14 wherein the blood sample is prepared by a method including an anion reduction step.

16. The method of claim 15 wherein the anion reduction step comprises a dilution step.

17. The method of claim 15 wherein the anion reduction step comprises a precipitation step.

18. The method of claim 1 wherein the sample comprises a plasma sample.

19. The method of claim 18 wherein the plasma sample is diluted from about 1:

2 to about 1;

15 plasma;

buffer.

20. The method of claim 18 wherein the plasma sample is diluted from about 1:

5 to about 1;

10 plasma;

buffer.

21. The method of claim 1 wherein the sample is a reconstituted dried blood spot sample.

22. The method of claim 21 wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 150 μ

L.

23. The method of claim 21 wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 100 μ

L.

24. The method of claim 21 wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 25 to about 75 μ

L.

25. The method of claim 21 wherein the reconstituted blood sample is reconstituted from a dried blood spot using an extraction volume ranging from about 40 to about 60 μ

L.

26. The method of claim 21 wherein the reconstituted blood sample is reconstituted from a dried blood spot using a buffer doped with surfactant.

27. The method of claim 26 wherein the surfactant comprises a polysorbate surfactant.

28. The method of claim 1, further comprising quantifying released 4-methylumbelliferone or an analog or derivative thereof.

29. The method of claim 1 wherein the reaction is performed at a temperature ranging from about 25 to about 40°

C.

30. The method of claim 1 wherein the reaction is performed at a temperature ranging from about 30 to about 40°

C.

31. The method of claim 1 wherein the reaction is performed at a temperature ranging from about 36 to about 39°

C.