Sucrose detection by enzyme-linked immunosorbant assay

• US 5,972,631 A
• Filed: 11/03/1997
• Issued: 10/26/1999
• Est. Priority Date: 11/03/1997
• Status: Expired due to Fees

• Current Assignee: De Novo Corporation
• Original Assignee: De NOVO Enzyme Corporation
• Inventors: Racher, Kathleen Iris, Borgford, Thor Jon, Braun, Curtis Archie John
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): NGUYEN, BAO THUY L
• Application Number:
  US08/962,723
• Time in Patent Office: 722 Days
• Field of Search: 435/14, 435/15, 435/18, 435/178, 435/210, 435/7.9, 435/7.32, 435/7.1, 435/7.94, 436/518, 436/524
• US Class Current: 435/7.9
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose

Abstract

A method is described for the rapid, sensitive and accurate determination of sucrose in biological fluids. A substrate is pre-coated with a glucose or fructose polymer and a transglycosidase enzyme. When the coated substrate is incubated with biological fluids containing concentrations of sucrose, the transglycosidase enzyme transfers monomers of glucose or fructose from the sucrose to the glucose or fructose polymer. The dimensions of the polymer are increased in proportion to the sucrose concentration of the samples. Newly formed polymer is subsequently quantitated in an immunoassay which employs either a combination of a carbohydrate-binding protein (which may be an antibody) and a conjugate of a secondary antibody and a marker enzyme, or a conjugate of a carbohydrate-binding protein and a marker enzyme. The assay is accurate at sucrose concentrations below 1 μg/mL. No interference was observed at glucose concentrations as high as 25 mM. The sucrose detection assay is particularly useful in a non-invasive diagnostic test for gastric damage.

Claims

1. A method for assaying sucrose in a biological fluid, said method comprising the steps of:

(a) coating a substrate with (i) a glucose or fructose polymer selected from the group consisting of amylose, dextran and levan and (ii) a sucrose transgylcosidase enzyme corresponding to said selected glucose or fructose polymer;

(b) incubating the coated substrate with a biological fluid; and

(c) determining an amount of sucrose present in the fluid by measuring an increase in an amount of the glucose or fructose polymer;

wherein step (c) comprises the steps of;

(i) incubating said substrate with a carbohydrate-binding protein, said protein being an anti-dextran antibody if the substate is coated with dextran, an amylose-binding protein if the substrate is coated with amylose, or an anti-levan antibody if the substrate is coated with levan;

(ii) incubating said substrate with a secondary antibody; and

(iii) adding to said substrate a solution containing a detecting agent, and determining the amount of sucrose in the fluid by measuring light absorption of the solution.

2. The method of claim 1, wherein the secondary antibody is a conjugate comprising (i) an antibody which will bind to the anti-dextran antibody, amylose-binding protein or anti-levan antibody and (ii) a marker enzyme.

4. The method of claims 2 or 3, wherein the marker enzyme is a peroxidase enzyme.

5. The method of claim 4, wherein the peroxidase enzyme is horseradish peroxidase.

6. The method of claim 5, wherein the detecting agent comprises hydrogen peroxide and a compound which oxidizes to form a chromophore which strongly absorbs visible light in amounts effective to produce a solution having a detectable absorption of visible light.

7. The method of claim 6, wherein the glucose or fructose polymer is dextran.

8. The method of claim 7, wherein the sucrose transglycosidase enzyme is dextransucrase.

9. The method of claim 8, wherein the carbohydrate-binding protein is mouse anti-dextran antibody.

10. The method of claim 9, wherein the detecting agent comprises hydrogen peroxide and o-phenylenediamine.

11. The method of claim 10, wherein the sucrose level is determined from a measurement of light absorption at 490 nm.

12. The method of claim 11, wherein the sucrose level is determined from a difference between a measurement of light absorption at about 490 nm and a measurement of light absorption at about 630 nm.

13. The method of claim 2, wherein the conjugate comprises goat anti-mouse IgG and horseradish peroxidase.

3. A method for assaying sucrose in a biological fluid, said method comprising the steps of:

(a) coating a substrate with (i) a glucose or fructose polymer selected from the group consisting of amylose, dextran and levan and (ii) a sucrose transgylcosidase enzyme corresponding to said selected glucose or fructose polymer;

(b) incubating the coated substrate with a biological fluid; and

(c) determining an amount of sucrose present in the fluid by measuring an increase in an amount of the glucose or fructose polymer;

wherein step (c) comprises the steps of;

(i) incubating said substrate with a conjugate comprising (1) a carbohydrate-binding protein, said protein being an anti-dextran antibody if the substate is coated with dextran, an amylose-binding protein if the substrate is coated with amylose, or an anti-levan antibody if the substrate is coated with levan; and

(2) a marker enzyme; and

(ii) adding to said substrate a solution containing a detecting agent, and determining the amount of sucrose in the fluid by measuring light absorption of the solution.