Assay for measuring enzymatic modification of a substrate by a glycoprotein having enzymatic activity

• US 9,140,635 B2
• Filed: 05/09/2012
• Issued: 09/22/2015
• Est. Priority Date: 05/10/2011
• Status: Expired due to Fees

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Graham, Carrie, Eckhardt, Allen, Perkins, Lisa
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US14/116,544
• Publication Number:
  US 20150050664A1
• Time in Patent Office: 1,231 Days
• Field of Search: 356/411
• US Class Current: 1/1
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 301/06013 Iduronate-2-sulfatase (3.1.6.13)
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01076 L-Iduronidase (3.2.1.76)
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: G01N 2333/928 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 2800/38 Pediatrics
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G16B 99/00 Subject matter not provided for in other groups of this subclass

Abstract

Methods of conducting an assay for measuring enzymatic modification of a substrate by a glycoprotein having enzymatic activity are provided. The methods include concentrating the glycoprotein having enzymatic activity by capturing the glycoproteins from the input sample on a solid support, washing the glycoproteins captured on a solid support to remove unbound portions of the input sample, adding an enzyme substrate to the output sample comprising glycoproteins having enzymatic activity, and measuring enzymatic modification of the substrate. In some examples, the capturing of the glycoprotein and subsequent washing steps are performed in one or more droplets in oil.

Claims

1. A method of conducting an assay for measuring enzymatic modification of the substrate by a glycoprotein having enzymatic activity, the method comprising:

(a) preparing an output sample comprising glycoproteins having enzymatic activity, comprising;

(i) providing an input sample comprising glycoproteins having enzymatic activity;

(ii) capturing the glycoproteins from the input sample on a solid support;

(iii) washing the glycoproteins captured on a solid support to remove unbound portions of the input sample, wherein steps (ii) and (iii) are performed in one or more droplets in oil; and

(iv) eluting glycoproteins from the solid support to yield an output sample comprising glycoproteins having enzymatic activity;

(b) adding an enzyme substrate to the output sample comprising glycoproteins having enzymatic activity; and

(c) measuring enzymatic modification of the substrate.

2. The method of claim 1, wherein the substrate comprises a glycoside substrate.

3. The method of claim 1, wherein the substrate releases a detectable fluorophore upon contact with the output sample comprising glycoproteins having enzymatic activity.

4. The method of claim 1, wherein the substrate comprises a glycoside substrate which releases a fluorophore upon contact with the output sample comprising glycoproteins having enzymatic activity.

5. The method claim 1, wherein the eluting is accomplished by contacting the solid support with one or more glycomimetics.

6. The method of claim 1, wherein the eluting is accomplished by cleaving a cleavable bond between the solid support and captured glycoproteins.

7. The method claim 1, wherein the input sample is selected from the group consisting of:

blood, plasma, serum, tears, saliva, and urine.

8. The method of claim 1, wherein the input sample comprises fresh blood.

9. The method of claim 1, wherein the input sample comprises reconstituted dried blood.

10. The method of claim 1, wherein the input sample consists essentially of plasma.

11. The method of claim 1, wherein the input sample comprises a human clinical sample.

12. The method of claim 1, wherein the solid support comprises concanavalin A.

13. The method of claim 1, wherein the solid support comprises a magnetically responsive bead.

14. The method of claim 1, wherein the captured glycoproteins comprise one or more enzymes.

15. The method of claim 1, wherein the captured glycoproteins comprise one or more glycosidase enzymes.

16. The method of claim 1, wherein the input sample is collected from a subject and immediately loaded onto a droplet actuator and the method is immediately performed.