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

• US 8,497,076 B2
• Filed: 04/26/2012
• Issued: 07/30/2013
• 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:
  US13/457,200
• Publication Number:
  US 20120208217A1
• Time in Patent Office: 460 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 pre-diabetes or diabetes, comprising:

determining a first glycosylation profile of a first sample from the subject, wherein the first glycosylation profile comprises one or more parameters relating to glycosylation; and

comparing the first glycosylation profile to a second glycosylation profile of a second sample from a control subject, wherein the second glycosylation profile comprises one or more parameters relating to glycosylation, wherein the control subject does not have pre-diabetes or diabetes;

wherein determining the glycosylation profile of the first and second samples comprises measuring binding of Aleuria aurantia lectin, Datura stramonium lectin, Phaseolus vulgaris agglutinin lectin, Concanavalin A, and Sambucus nigra lectin to the first and second samples; and

wherein an increase in binding of Aleuria aurantia lectin, Datura stramonium lectin, and Phaseolus vulgaris agglutinin lectin to the first sample relative to the second sample indicates that the subject has either pre-diabetes or diabetes; and

wherein an increase in binding of Aleuria aurantia lectin, Datura stramonium lectin, Phaseolus vulgaris agglutinin lectin, Concanavalin A, and Sambucus nigra lectin to the first sample relative to the second sample indicates that the subject has diabetes, and not pre-diabetes.

2. The method of claim 1, wherein both of the first and second samples comprise urine, saliva, or serum.

3. The method of claim 1, wherein an increase in Concanavalin A and/or Sambucus nigra lectin binding to the first sample relative to the second sample indicates the subject has diabetes.

4. 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 an increase 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 control subject indicates that the subject has pre-diabetes or diabetes.

5. The method of claim 4, wherein determining the quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein comprises performing an immunoassay.

6. The method of claim 1, further comprising:

determining a glycosylation state of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the first sample; and

comparing the glycosylation state of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the first sample to the glycosylation state of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the second sample, wherein a difference in the glycosylation state of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the first sample as compared to the glycosylation state of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the second sample indicates that the subject has pre-diabetes or diabetes.

7. The method of claim 6, wherein determining the glycosylation state of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein comprises detecting and/or measuring lectin binding to the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein.

8. The method of claim 7, wherein determining the glycosylation profile of the first and second samples further comprises measuring binding of Concanavalin A and Sambucus nigra lectin:

and wherein one or more of the Aleuria aurantia lectin, Datura stramonium lectin, Phaseolus vulgaris agglutinin lectin, Concanavalin A, or Sambucus nigra lectin bind alpha-1 acid glycoprotein.

9. The method of claim 6, further comprising determining a quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein in the first and second samples.

10. The method of claim 9, wherein an increase in the quantity of alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) in the first sample as compared to the second sample indicates that the subject has pre-diabetes or diabetes.

11. The method of claim 9, wherein determining the quantity of the alpha-1 acid glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein comprises performing an immunoassay.

12. The method of claim 1, wherein determining the glycosylation profile of the first sample comprises contacting a the first sample with a lateral flow device comprising the Aleuria aurantia lectin, Datura stramonium lectin, and Phaseolus vulgaris agglutinin lectin.

13. The method of claim 1, wherein an increase in blinding of the Aleuria aurantia lectin, Datura stramonium lectin, or Phaseolus vulgaris agglutinin lectin to the first sample relative to the second sample indicates that the subject has a diabetic complication.

14. The method of claim 13, wherein the diabetic complication is a microvascular complication or a macrovascular complication.

15. The method of claim 14, wherein the diabetic complication is a microvascular complication comprising one or more of diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy.