Methods for detecting pre-diabetes and diabetes using differential protein glycosylation

• US 8,168,396 B2
• Filed: 05/11/2010
• Issued: 05/01/2012
• Est. Priority Date: 05/11/2009
• Status: Active Grant

• Current Assignee: DiabetOmics, Inc.
• Original Assignee: Diabetomics, LLC
• Inventors: Nagalla, Srinivasa R., Roberts, Charles T.
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US12/777,955
• Publication Number:
  US 20100285498A1
• Time in Patent Office: 721 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2333/42 Lectins, e.g. concanavalin, phytohaemagglutinin
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 2440/38 addition of carbohydrates, e.g. glycosylation, glycation
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 2800/50 Determining the risk of developing a disease
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Methods for identifying individuals who are not yet diabetic (pre-diabetic), but who are at significant risk of developing diabetes, such as type 2 diabetes, are disclosed herein. Methods are also provided for the identification of diabetic subjects. Also disclosed are methods for identifying individuals with diabetic complications. The methods include the identification of an overall glycosylation profile of proteins in a biological fluid, such as saliva, urine, or serum. In some examples, the methods include determining the amount of one or more protein in a biological fluid or determining the glycosylation pattern of one or more proteins in a biological fluid.

Claims

1. A method for identifying a subject with a metabolic condition, comprising:

determining a glycosylation profile of a sample from the subject, wherein the glycosylation profile is determined using a ratio of lectin binding; and

comparing the glycosylation profile to the glycosylation profile of a sample from a normal control subject, wherein a difference in the glycosylation profile of the sample from the subject as compared to the sample from the normal control subject indicates that the subject has a metabolic condition.

2. The method of claim 1, wherein the metabolic condition is pre-diabetes or diabetes.

3. The method of claim 1, wherein the sample comprises urine, saliva, or serum.

4. The method of claim 1, wherein an increase in a parameter of the glycosylation profile indicates the subject has pre-diabetes or diabetes.

5. The method of claim 1, wherein determining the glycosylation profile of the sample is determined using lectin binding, wherein increased lectin binding indicates that the subject has pre-diabetes or diabetes.

6. The method of claim 5, wherein the lectin is selected from the group consisting of Aleuria aurantia lectin, Concanavalin A, Datura stramonium lectin, Erythrina cristagalli lectin, Lycopersicon esculentum lectin, Maackia amurensis lectin I, Phaseolus vulgaris agglutinin, and Sambucus nigra lectin.

7. The method of claim 5, wherein increased lectin binding by Aleuria aurantia lectin, Datura stramonium lectin, or Phaseolus vulgaris agglutinin indicates the subject has pre-diabetes.

8. The method of claim 5, wherein increased lectin binding by Concanavalin A or Sambucus nigra lectin indicates the subject has diabetes.

9. The method of claim 1, wherein the ratio of lectin binding is a ratio of Sambucus nigra lectin to Maackia amurensis lectin I, a ratio of Concanavalin A to Maackia amurensis lectin I, or a ratio Concanavalin A to Aleuria aurantia lectin.

10. The method of claim 9, wherein an increase in the ratio of Sambucus nigra lectin to Maackia amurensis lectin I or an increase in the ratio of Concanavalin A to Maackia amurensis lectin I indicates the subject has diabetes.

11. The method of claim 1, further comprising determining a quantity of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the sample from the subject, wherein a difference in the quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the sample from the subject as compared to the sample from the normal control subject indicates that the subject has pre-diabetes or diabetes.

12. The method of claim 11, wherein an increase in the quantity of A1AG, A1AT, a ratio of A1AG to A1AT, or a combination thereof, as compared to the control sample indicates that the subject has pre-diabetes or diabetes.

13. The method of claim 11, wherein determining the quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein comprises an immunoassay.

14. The method of claim 1, further comprising:

determining a glycosylation pattern of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) in the sample from the subject; and

comparing the glycosylation pattern of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) in the sample to the glycosylation pattern of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) in a sample from a normal control subject, wherein a difference in the glycosylation profile or glycosylation pattern in the sample from the subject as compared to the sample from the normal control subject indicates that the subject has pre-diabetes or diabetes.

15. The method of claim 14, wherein determining the glycosylation pattern of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) is determined using lectin binding, wherein increased lectin binding indicates that the subject has pre-diabetes or diabetes.

16. The method of claim 15, wherein the lectin comprises one or more lectins that bind alpha-1 acid glycoprotein.

17. The method of claim 16, wherein the lectin comprises one of more of Concanavalin A, Aleuria aurantia lectin, or Sambucus nigra lectin.

18. The method of claim 17, wherein increased lectin binding by Concanavalin A or Sambucus nigra lectin indicates the subject has pre-diabetes.

19. The method of claim 14, further comprising determining a quantity of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the sample from the subject.

20. The method of claim 19, wherein an increase in the quantity of A1AG, A1AT, a ratio of A1AG to A1AT, or a combination thereof, as compared to the control sample indicates that the subject has pre-diabetes or diabetes.

21. The method of claim 19, wherein determining the quantity of the alpha-1 acid glycoprotein A1AG and/or alpha-1 antitrypsin (A1AT) protein comprises an immunoassay.

22. The method of claim 1, wherein determining a glycosylation profile of a sample from the subject comprises contacting a sample from the subject with a lateral flow device comprising one or more lectins.

23. The method of claim 22, wherein the one or more lectins is selected from the group consisting of Aleuria aurantia lectin, Concanavalin A, Datura stramonium lectin, Erythrina cristagalli lectin, Lycopersicon esculentum lectin, Maackia amurensis lectin I, Phaseolus vulgaris agglutinin, and Sambucus nigra lectin.

24. The method of claim 1, wherein the metabolic condition is a diabetic complication.

25. The method of claim 24, wherein the diabetic complication is a microvascular complication or a macrovascular complication.

26. The method of claim 25, wherein the microvascular complication comprises one or more or diabetic nephropathy, diabetic retinopathy, or diabetic neuropathy.

27. The method of claim 24, wherein the glycosylation profile is determined using lectin binding, wherein increased lectin binding indicates that the subject has the diabetic complication.

28. The method of claim 27, wherein increased lectin binding by Aleuria aurantia lectin or Sambucus nigra lectin indicates the subject has the diabetic complication.

29. The method of claim 24, further comprising determining a quantity of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the sample from the subject, wherein a difference in the quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the sample from the subject as compared to the sample from the normal control subject indicates that the subject has a diabetic complication.