WO2010011860A1

WO2010011860A1 - Methods for detecting pre-diabetes and diabetes

Info

Publication number: WO2010011860A1
Application number: PCT/US2009/051578
Authority: WO
Inventors: Srinivasa R. Nagalla; Charles T. Roberts; Vishnupriya Rao Paturi
Original assignee: Diabetomics, Llc
Priority date: 2008-07-23
Filing date: 2009-07-23
Publication date: 2010-01-28
Also published as: US8476008B2; US20110124022A1

Abstract

Non-invasive methods are provided herein for the diagnosis of pre-diabetes and diabetes using biomarkers identified in a biological fluid, such as saliva. These biomarkers can be identified using proteomic methods, including but not limited to antibody based methods, such as an enzyme-linked immunosorbant assay (ELISA), a radioimmnoassay (RIA), or a lateral flow immunoassay.

Description

METHODS FOR DETECTING PRE DIABETES AND DIABETES

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/083,096, filed July 23, 2008, which is incorporated herein in its entirety.

FIELD

This relates to the field of diabetes, specifically to the identification of subjects who have diabetes or pre-diabetes, who are at risk of developing diabetes or pre-diabetes and/or to monitoring the effectiveness of treatments for diabetes.

BACKGROUND

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism that result from defects in insulin secretion, insulin action, or both. Diabetes can present with characteristic symptoms such as thirst, polyuria, blurring of vision, chronic infections, slow wound healing, and weight loss. In its most severe forms, ketoacidosis or a non-ketotic hyperosmolar state may develop and lead to stupor, coma and, in the absence of effective treatment, death. Diabetes mellitus is subdivided into type 1 diabetes and type 2 diabetes.

Type 1 diabetes results from autoimmune mediated destruction of the beta cells of the pancreas. Patients with type 1 diabetes exhibit little or no insulin secretion as manifested by low or undetectable levels of insulin or plasma C-peptide (also known in the art as "soluble C-peptide"). Type 2 diabetes is characterized by disorders of insulin action and insulin secretion, either of which may be the predominant feature. Type 2 diabetes patients can be both insulin deficient and insulin resistant. At least initially, and often throughout their lifetime, these individuals do not need supplemental insulin treatment to survive. Type 2 diabetes accounts for 90-95% of all cases of diabetes and can go undiagnosed for many years because the hyperglycemia is often not severe enough to provoke noticeable symptoms of diabetes or symptoms are simply not recognized. The majority of patients with type 2 diabetes are obese, and obesity itself may cause or aggravate insulin resistance. Many of those who are not obese by traditional weight criteria may have an increased percentage of body fat distributed predominantly in the abdominal region (visceral fat). The symptoms of the early stages of diabetes often are not severe, not recognized, or may be absent. Consequently, hyperglycemia sufficient to cause pathological and functional changes may be present for a long time, occasionally up to ten years, before a diagnosis is made, usually by the detection of high levels of glucose in urine after overnight fasting during a routine medical work-up. The long- term effects of diabetes include progressive development of complications such as retinopathy with potential blindness, nephropathy that may lead to renal failure, neuropathy, microvascular changes, and autonomic dysfunction. People with diabetes are also at increased risk of cardiovascular, peripheral vascular, and cerebrovascular disease, as well as an increased risk of cancer. Several pathogenic processes are involved in the development of diabetes, including processes which destroy the insulin-secreting beta cells of the pancreas with consequent insulin deficiency, and changes in liver and smooth muscle cells that result in the resistance to insulin uptake. The abnormalities of carbohydrate, fat and protein metabolism are due to deficient action of insulin on target tissues resulting from insensitivity to insulin (insulin resistance) or lack of insulin (loss of beta cell function).

Over 18 million people in the United States have type 2 diabetes, and of these, about 5 million do not know they have the disease. These persons, who do not know they have the disease and who do not exhibit the classic symptoms of diabetes, present a major diagnostic and therapeutic challenge. Nearly 41 million persons in the United States are at significant risk of developing type 2 diabetes. These persons are broadly referred to as "pre-diabetics." The risk of developing type 2 diabetes increases with age, obesity, and lack of physical activity. It occurs more frequently in women with prior gestational diabetes, and in individuals with hypertension and/or dyslipidemia. As intervention early in the development of diabetes can substantially affect the long-term prognosis of the disease, a need remains to identify individuals who are pre-diabetic or those subjects who will become diabetic. In addition, a need remains for monitoring therapeutic interventions, to determine if they are effective.

SUMMARY Pre-diabetes can be present for ten or more years before the detection of glycemic abnormalities and the development of actual diabetes. Treatment of pre- diabetics with drugs such as acarbose, metformin, troglitazone and rosiglitazone can postpone or prevent diabetes; yet few pre-diabetics are treated. A major reason is that no simple and unambiguous laboratory test has existed that can be used to identify those subjects at risk for developing diabetes or pre-diabetes. In addition, there is a need for a test that can determine the effectiveness of therapy for prediabetes and/or diabetes. There also is a need to identify subjects with a diabetic condition, including both pre-diabetic and diabetic subjects, so that they can obtain treatment early, and also to monitor the progression of the disease over time non- invasively.

Methods for identifying individuals who are not yet diabetics (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. These methods can be used to select subjects for therapeutic or lifestyle intervention. In additional embodiments, the methods can be used to identify diabetes type 2 in a subject (as compared to pre-diabetes). In further embodiments, the methods can be used to identify the efficacy of a therapeutic intervention, such as to manage dosage over time of anti-diabetic medication, or to asses the success of lifestyle interventions or other treatments to modify disease progression. The methods include the identification of biomarkers such as proteins in a biological fluid, such as saliva. In some embodiments, these biomarkers are identified using antibody-based methods, such as, but not limited to, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), a lateral flow immunoassay, or proteomic approaches that utilize various detection methods. In several embodiments, methods are provided for the diagnosis of prediabetes, determining if a subject is at risk for developing pre-diabetes, or monitoring the efficacy of therapy, including lifestyle modifications and preventative treatments, in a human subject of interest. The methods include testing in a biological sample (such as saliva) obtained from said subject the abundance (amount) of one or more proteins relative to the abundance (amount) in a biological sample (such as saliva) from a pre-diabetic subject or a control subject. In some examples, the control is a sample from a subject not known to have impaired glucose tolerance, impaired fasting glucose regulation, or both. The subject of interest is diagnosed with pre-diabetes or diabetes, determined to be at risk for developing prediabetes, or having an effective therapeutic regimen if the abundance (amount) is not statistically significantly different relative to abundance in the saliva from the pre- diabetic or diabetic subject, respectively, or is statistically increased relative to abundance in a saliva sample from the control subject.

The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a pie chart showing the functional annotation of type 2 diabetes human whole saliva proteome. Salivary proteins were functionally annotated using DAVID™ and BioHarvester informatics resources. The majority of the proteins are predicted to have metabolic and immune response functions.

FIG. 2A is a digital image of a Western blot analysis of alpha-2 macroglobulin (A2MG), alpha- 1 -antitrypsin (AlAT), cystatin C (Cys C), Transthyretin (TTR), and salivary alpha-amylase (AMYS).

FIG. 2B is a bar graph showing ELISA analysis of saliva A2MG showing mean concentrations in controls (CTRL) and study subjects with impaired glucose tolerance (IGT), IGT and impaired fasting glucose (IFG), and diabetes mellitus (DM). Error bars denote SEM. p=0.0186 for the 4-group comparison via Kruskal- Wallis nonparametric ANOVA. p=0.0137 for pair. FIG. 3A is a perspective view of a physical embodiment of a lateral flow test strip showing the basic components of the device and their relationship to each other.

FIG. 3B is a perspective view of a physical embodiment of a lateral flow test strip for the detection of multiple analytes.

FIG. 4 is a perspective view of a physical embodiment of an exemplary lateral flow test strip for diagnosing pre-diabetes or diabetes utilizing a sandwich immunoassay format.

FIG. 5 is a perspective view of a physical embodiment of an exemplary lateral flow test strip for diagnosing pre-diabetes or diabetes utilizing a competitive immunoassay format.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R.

1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. SEQ ID NOs: 1-67 show exemplary amino acid sequences of salivary proteome proteins differentially abundant in pre-diabetes and diabetes relative to normal.

DETAILED DESCRIPTION The twin epidemics of obesity and diabetes threaten to overwhelm healthcare systems in the US and worldwide (Smyth and Heron, Nat Med 12:75-80, 2005). Over the last several years, the number of persons in the United States diagnosed with diabetes has reached almost epidemic proportions, with about 18 million affected individuals in the United States alone, at a cost of 174 billion dollars for 2007 (American Diabetes Association, Diabetes Care 31:596-615, 2008). Improved detection techniques and biomarkers are urgently needed across the entire spectrum of diabetes initiation and progression. Since 70% of pre-diabetics will progress to frank diabetes (Nathan et al., Diabetes Care 30:753-759, 2007), and 7% of newly diagnosed type 2 diabetes mellitus patients in the US have been diabetic for approximately 4 to 7 years before diagnosis (Harris et al., Diabetes Care 15:815-819, 1992), the ability to ascertain those individuals at risk for the development of clinically apparent diabetes is critical to effectively focus potentially limited clinical resources. In particular, it is desirable to screen and start treating glucose-intolerant individuals as early as possible since, even before the onset of diabetes, vascular lesions gradually develop with deterioration of glucose tolerance. Additionally, beta-cell function is seriously compromised by the time that overt alterations in glucose homeostasis, such as impaired glucose tolerance (IGT) and impaired fasting glucose (IFG), are manifest; thus, timely intervention is important to maintain residual insulin secretory capacity.

The effectiveness of early intervention with lifestyle modification or medication in arresting disease progression has been demonstrated by the Diabetes Prevention Program (Diabetes Prevention Program Research Group. NEJM 346:393-403, 2002). However, the determination of IGT and IFG is itself an issue due to the relatively invasive nature of these assessments, particularly that of IGT by an oral glucose tolerance test (OGTT). In addition, an important additional diagnostic problem is monitoring of glucose homeostasis for confirming diabetes. Compliance with glucose monitoring is poor because of the pain and inconvenience of conventional blood collection using lancets. Furthermore, non- invasive monitoring techniques for diabetes, and to determine the efficacy of therapy, are desirable. Finally, assessment of progression of frank diabetes to complications is only feasible after complications are well established. Thus, it would be beneficial to have methods for assessing the development of diabetes from pre-diabetes, and for monitoring the course of the disease.

Saliva has a number of advantages as a diagnostic fluid. These include: noninvasive collection; feasible without special training or equipment; especially advantageous for pediatric or elderly populations; and amenable to large-scale population studies. The salivary proteome is derived from a number of sources, including major and minor salivary gland secretions, oral bacterial products, and gingival crevicular fluid (GCF) (Humphrey et al., J Pros Dent 85:162-169, 2001). Although the majority of salivary protein by amount is comprised of the major classes of salivary protein families such as the acidic and basic proline-rich proteins, amylase, and various mucins, the salivary proteome as a whole is dynamic and complex (for discussion, see Helmerhorst and Oppenheim, Crit Rev Oral Biol Med 680-693, 2007). Of particular interest is GCF, which is considered a transudate or ultrafiltrate of serum. Although the proportional contribution of GCF to saliva is small, it allows saliva to exhibit levels of serum-derived proteins that may reflect their circulating levels.

Thus, saliva can be used to determine the effect of a treatment protocol or a disease process in an individual of interest. For example, it is possible to measure proteins such as insulin and GIP (Messenger et al., J Endocrinol 177:407-412, 2003), prolactin (Huang, Arch Oral Biol 49:951-962, 2004) and GH (Rantonen et al., Acta Odontol Scand 58:299-303, 2000) in saliva. Previous studies have described the salivary proteome (Yamguchi et al., Biomed Microdev 7:53-58, 2005; see also, for example, Hu et al., Proteomics 5:1714-1728, 2005) while other studies have described alterations in salivary dynamics (Dodds et al., Community Dent Oral Epidemiol 28:373-381, 2000; Bernardi et al., Oral Health Prev Dent 5:73-78, 2007) or the differential abundance in saliva of single factors, such as MMP-8 (Collin et al., J Periodontal Res 35:259-265, 2000) and EGF (Oxford et al., J Diabetes Complications 14:140-145, 2000).

Presented below is the first comprehensive analysis of the salivary proteome in diabetes. In some embodiments of the methods disclosed herein, this proteome can be used to diagnose diabetes and pre-diabetes, as well as to monitor the progression of the disease and to assess the efficacy of a particular therapeutic intervention. Thus, non-invasive methods are provided herein for the diagnosis of pre-diabetes and diabetes using biomarkers identified in a biological fluid, such as saliva. Non-invasive methods are also provided to identify those subjects at risk of developing pre-diabetes and diabetes using these biomarkers. Additionally, noninvasive methods are provided for evaluating the efficacy of a therapy using the biomarkers. These biomarkers also can be identified using antibody-based methods, such as, but not limited to, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or lateral flow immunoassay, and other proteomic approaches with or without use of antibodies

Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). All the amino acid sequences identified herein, as set forth in GENB ANK® on March 10, 2008, are incorporated by reference herein. This includes all those shown in the tables, as well as described in the examples section.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Alpha- 1-antitrypsin: A 52 kDa serine protease inhibitor that is considered the most prominent serpin. The protein was called "antitrypsin" because of its ability to covalently bind and irreversibly inactivate the enzyme trypsin in vitro. The term alpha-1 refers to the enzyme's behavior on protein electrophoresis. There are several "clusters" of proteins in electrophoresis, the first being albumin, the second being the alpha, the third beta and the fourth gamma (immunoglobulins). The non- albumin proteins are referred to as globulins. The alpha region can be further divided into two sub-regions, termed "1" and "2". Alpha 1-antitrypsin is the main enzyme of the alpha-globulin 1 region. An exemplary amino acid sequence is shown in GENB ANK® Accession No. P01009 (March 10, 2008), incorporated by reference herein. Alpha-2-macroglobulin: A large plasma protein found in the blood. It is produced by the liver, and is a major component of the alpha-2 band in protein electrophoresis. Alpha-2 macroglobulin is able to inactivate an enormous variety of proteinases (including serine-, cysteine-, aspartic- and metalloproteinases). Alpha-2 macroglobulin has in its structure a 35 amino acid "bait" region. Proteinases binding and cleaving the bait region become bound to α2M. The proteinase- α2M complex is recognized by macrophage receptors and cleared from the system. It functions as an inhibitor of coagulation by inhibiting thrombin and it functions as an inhibitor of fibrinolysis by inhibiting plasmin. An exemplary amino acid sequence is shown in GENB ANK® Accession No. P01023 (March 10, 2008), incorporated by reference herein. Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example mice.

Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically binds an epitope of a protein listed in the tables below, or a fragment of any of these proteins. The term "specifically binds" refers to, with respect to an antigen such the proteins listed in the tables below, the preferential association of an antibody or other ligand, in whole or part, with the protein. A specific binding agent binds substantially only to a defined target, such as protein of interest. Thus, as a non- limiting example, an alpha- 1 -antitrypsin specific binding agent is an agent that binds substantially to an alpha- 1 -antitrypsin polypeptide. If an agent, such as an antibody, specifically binds alpha- 1 -antitrypsin it does not specifically bind other peptides including cystatin C, alpha-2-macroglobulin or transthyretin, or any of the other proteins listed in the tables below. A minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Specific binding can be distinguished as mediated through specific recognition of the antigen.

A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

Antibodies can include a heavy chain and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A "monoclonal antibody" is an antibody produced by a single clone of B -lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies. Anti-diabetic agent: A chemical or pharmaceutical anti-hyperglycemic agent or drug capable of treating diabetes, including, but not limited to agents for alleviating the symptoms associated with type 2 diabetes or slowing the progression or onset of type 2 diabetes. Anti-diabetic agents are generally categorized into six classes: biguanides; thiazolidinediones; sulfonylureas; inhibitors of carbohydrate absorption; fatty acid oxidase inhibitors and anti-lipolytic drugs; and weight-loss agents. The anti-diabetic agents include those agents disclosed in Diabetes Care, 22(4):623-34, herein incorporated by reference. One common class of anti-diabetic agents is the sulfonylureas, which are believed to increase secretion of insulin, decrease hepatic glucogenesis, and increase insulin receptor sensitivity.

Another class of anti-diabetic agents is the biguanide antihyperglycemics, which decrease hepatic glucose production and intestinal absorption, and increase peripheral glucose uptake and utilization, without inducing hyperinsulinemia. The biguanide anti-diabetic agents include compounds defined by the chemical formula of Formula 1 (see below), such as the biguanides disclosed in U.S. Pat. Nos. 3,960,949; 4,017,539; and 6,011,049, herein incorporated by reference. One specific, non-limiting example of a biguanide antidiabetic agent is metformin.

Incretins are another class of anti-diabetic agents. These agents are described in further detail below.

Anti-diabetic lifestyle modifications: Changes to lifestyle, habits, and practices intended to alleviate the symptoms of diabetes or pre-diabetes. Obesity and sedentary lifestyle may both independently increase the risk of a subject developing type II diabetes, so anti-diabetic lifestyle modifications include those changes that will lead to a reduction in a subject's body mass index (BMI), increase physical activity, or both. Specific, non-limiting examples include the lifestyle interventions described in Diabetes Care, 22(4):623-34 at pages 626-27, herein incorporated by reference.

Binding: A specific interaction between two or more molecules, such as the binding of an antibody and an antigen (for example an antibody to an antigen). In one embodiment, specific binding is identified by a dissociation constant (Kd). In one embodiment, binding affinity is calculated by a modification of the Scatchard method described by Frankel et a , MoI. Immunol , 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay (RIA). In several examples, a high binding affinity is at least about 1 x 10^~8 M. In other embodiments, a high binding affinity is at least about 1.5 x 10^~8, at least about 2.0 x 10^~8, at least about 2.5 x 10^~8, at least about 3.0 x 10^"8, at least about 3.5 x 10^~8, at least about 4.0 x 10^~8, at least about 4.5 x 10^~8, or at least about 5.0 x 10^~8 M.

Body mass index (BMI): A mathematical formula for measuring body mass in humans, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height (in meters ). The current standards for both men and women accepted as "normal" are a BMI of 20-24.9 kg/m². In one embodiment, a BMI of greater than 25 kg/m can be used to identify an obese subject. Grade I obesity (also called "overweight") corresponds to a BMI of 25-29.9 kg/m². Grade II obesity corresponds to a BMI of 30-40 kg/m²; and Grade III obesity corresponds to a BMI greater than 40 kg/m² (Jequier, Am. J Clin. Nutr., 45:1035-47, 1987). Ideal body weight will vary among species and individuals based on height, body build, bone structure, and sex.

Chromatography: The process of separating a mixture, for example a mixture containing the proteins listed in the tables below. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows one or more molecules of interest to be isolated. Examples of methods of chromatographic separation include capillary-action chromatography, such as paper chromatography, thin layer chromatography (TLC), column chromatography, fast protein liquid chromatography (FPLC), nano-re versed phase liquid chromatography, ion exchange chromatography, gel chromatography, such as gel filtration chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and reverse phase high performance liquid chromatography (RP-HPLC) amongst others.

Contacting: "Contacting" includes in solution and solid phase, for example contacting a salivary protein with a test agent. The test agent may also be a combinatorial library for screening a plurality of compounds. In another example, contacting includes contacting a sample with an antibody, for example contacting a sample that contains a protein of interest such as those listed in the tables below. Cystatin-C: A serum protein used mainly as a measure of glomerular filtration rate. It is a single 120-residue polypeptide belonging to the type 2 cystatin gene family. Studies have shown that Cystatin C allows a more precise testing of kidney function than creatinine. The type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. The cystatin locus on chromosome 20 contains the majority of the type 2 cystatin genes and pseudogenes. This gene is located in the cystatin locus and encodes the most abundant extracellular inhibitor of cysteine proteases, which is found in high concentrations in biological fluids. An exemplary amino acid sequence is shown in GENB ANK® Accession No. PO 1034.

Diabetes mellitus: A disease caused by a relative or absolute lack of insulin leading to uncontrolled carbohydrate metabolism, commonly simplified to "diabetes," though diabetes mellitus should not be confused with diabetes insipidus. As used herein, "diabetes" refers to diabetes mellitus, unless otherwise indicated. A "diabetic condition" includes pre-diabetes and diabetes. Type 1 diabetes (sometimes referred to as "insulin dependent diabetes" or "juvenile onset diabetes") is an autoimmune disease characterized by destruction of the pancreatic β cells that leads to a total or near total lack of insulin. In diabetes type 2 (sometimes referred to as "non- insulin dependent diabetes" or "adult onset diabetes"), the body does not respond to insulin, though it is present.

Symptoms of diabetes include: excessive thirst (polydipsia); frequent urination (polyuria); extreme hunger or constant eating (polyphagia); unexplained weight loss; presence of glucose in the urine (glycosuria); tiredness or fatigue; changes in vision; numbness or tingling in the extremities (hands, feet); slow- healing wounds or sores; and abnormally high frequency of infection. Diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load. A more detailed description of diabetes may be found in Cecil Textbook of Medicine, J.B. Wyngaarden, et al., eds. (W.B. Saunders Co., Philadelphia, 1992, 19^th ed.).

A subject exhibiting one or more of the following risk factors is considered to have a heightened or substantial risk of developing diabetes type 2:

1. Obesity, such as a BMI greater than or equal to about 30 kg/m²; 2. Elevated fasting blood glucose (FPG) levels;

3. Impaired glucose tolerance (IGT);

4. Non-caucasian ethnicity;

5. Hyperinsulinemia; 6. Hypertriglyceridemia;

7. Family history of diabetes;

8. History of gestational diabetes;

9. Sedentary lifestyle;

10. In humans, middle age or elderly status (i.e., 40 years old and older). The methods disclosed herein provide a means of identifying s subject who has diabetes or pre-diabetes, or who is at increased risk of developing diabetes, including both type 1 and type 2 diabetes. A "non-diabetic" or "normal" subject does not have any form of diabetes, such as type 1 diabetes, type 2 diabetes, or prediabetes. Fasting plasma glucose (FPG): A diagnostic test for diabetes or prediabetes. The blood glucose concentration or level of a subject is analyzed in a blood sample obtained from a subject after the subject has fasted overnight or undergone a fast of at least 8 hours. A diabetic subject will often show a heightened blood glucose level, compared to a non-diabetic subject. Generally, a fasting plasma glucose test (FPG) is used to determine if a subject has impaired fasting glucose. An FPG of greater than 100 mg/dl and less than 126 mg/dl indicates that a subject has pre-diabetes. A FPG greater than or equal to 126 mg/dl indicates that a subject has frank diabetes, and an FPG of equal to or less than 100 mg/dl indices that subject is normal (healthy) and does not have pre-diabetes or diabetes. For example, the subject is not known to have diabetes type 2, and/or does not satisfy diagnostic criteria for diabetes type 2 and pre-diabetes.

Food intake: The amount of food consumed by an individual. Food intake can be measured by volume or by weight. In one embodiment, food intake is the total amount of food consumed by an individual. In another embodiment, food intake is the amount of proteins, fat, carbohydrates, cholesterol, vitamins, minerals, or any other food component, of the individual. "Protein intake" refers to the amount of protein consumed by an individual. Similarly, "fat intake," "carbohydrate intake," "cholesterol intake," "vitamin intake," and "mineral intake" refer to the amount of proteins, fat, carbohydrates, cholesterol, vitamins, or minerals consumed by an individual. Hyperglycemia: An above-normal level of glucose (sugar) in the blood, and an indicator of diabetes. Hyperglycemia occurs when the body either lacks sufficient insulin or cannot use available insulin to metabolize glucose. Symptoms of hyperglycemia include excessive thirst, a dry mouth, and frequent urination.

Isolated: An isolated biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example the separation of a peptide from a sample, such as saliva, serum or blood. Peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods, such as chromatography, for example high performance liquid chromatography (HPLC) and the like. The term also embraces peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptide and nucleic acids. It is understood that the term "isolated" does not imply that the biological component is free of trace contamination, and can include molecules that are at least 50% isolated, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or even 100% isolated.

Immunoassay: A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of antigen or the amount of antigen present can be measured. For measuring proteins, for each the antigen and the presence and amount (abundance) of the protein can determined or measured.

Measuring the quantity of antigen (such as any of the proteins listed in the Tables herein) can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody with a detectable label. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example C, P, I, and H isotopes and the like). In some examples alpha- 1 -antitrypsin, cystatin C, alpha-2-macroglobulin or transthyretin is labeled with a radioactive isotope, such as ¹⁴C, ³²P, ¹²⁵1, ³H isotope. In other examples an antibody that specifically binds one of an antigen of interest is labeled. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Harlow & Lane, (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988)),

A "competitive radioimmunoassay (RIA)" is a type of immunoassay used to test for antigens (for example, proteins present in a sample, such as a biological sample). In some examples it involves mixing known quantities of radioactive antigen (for example a radioactively labeled protein, such as a ¹²⁵I labeled protein) with antibody to that antigen, then adding unlabeled or "cold" antigen (for example unlabeled antigen present in a sample, such as biological sample obtained from a subject, such as saliva) and measuring the amount of labeled antigen displaced by the unlabeled antigen.

Initially, the radioactive antigen is bound to the antibodies. When "cold" (i.e. unlabeled) antigen is added, the two compete for antibody binding sites at higher concentrations of "cold" antigen, more of it binds to the antibody, displacing the radioactive variant. The bound antigens are isolated from the unbound ones and the amount of radioactivity measured. A radioimmunoassay can be used to calculate the amount of an antigen in a sample. Incretin: Gastrointestinal peptides that affect glycemic control, including amylin, gastric inhibitory peptide (GIP), and glucagon- like peptide 1 (GLP-I). During the past few years, analogs of these hormones have become available for use in controlling diabetes. One incretin, pramlintide, is an analog of amylin, a naturally occurring hormone produced along with insulin by pancreatic β-cells. Levels increase postprandially and typically correlate with insulin levels. As with insulin, amylin levels are very low in type 1 diabetes; however, levels may be elevated in patients with insulin resistance. Administration of exogenous amylin in the form of pramlintide has been shown to decrease postprandial hyperglycemia in patients with type 1 or type 2 diabetes who are treated with insulin. The major mechanism of action appears to be inhibition of gastric emptying and suppression of glucagon release. Clinically, it also suppresses the appetite in those who receive it.

Exenatide is an analog of GLP-I, a naturally occurring incretin produced by the L-cells of the distal ileum. GLP- 1 acts to stimulate insulin release from the pancreatic β-cells, suppress glucagon release from the pancreatic α-cells, slow gastric emptying, and increase satiety. Administration of exenatide in patients with type 2 diabetes has similar effects to pramlintide. Clinically, the result is a reduction in AlC of -1%. Preliminary studies suggest that a significant proportion of patients with type 2 diabetes using insulin may be successfully transitioned from insulin to exenatide in addition to their oral agents.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example ¹⁴C, ³²P, ¹²⁵1, ³H isotopes and the like). In some examples a protein, such as one of the proteins listed in the Tables herein, is labeled with a radioactive isotope, such as ¹⁴C, ³²P, ¹² 1, ³H isotope. In some examples an antibody that specifically binds the protein is labeled. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), Harlow & Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988).

Obesity: A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, National Heart, Lung, and Blood Institute (NHLBI), Obes. Res. 6 (suppl. 2):51S- 209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure. In one embodiment in humans, the Body Mass Index (BMI) is used to assess obesity. In one embodiment, a BMI of 25.0 kg/m² to 29.9 kg/m² is overweight (also called grade I obesity), while a BMI of 30 kg/m² is truly obese (also called grade II obesity).

In another embodiment in humans, waist circumference is used to assess obesity. In this embodiment, in men a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese. Strong evidence shows that obesity affects both the morbidity and mortality of individuals. For example, an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer (e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et al., Am. Fam. Phys. 63:2185, 2001).

Oral glucose tolerance test (OGTT): A diagnostic test for diabetes. After fasting overnight, a subject is provided a concentrated sugar solution to drink, usually containing 50 to 100 grams of glucose. The subject's blood is sampled periodically over the next few to several hours to test blood glucose levels over time. In a non-diabetic subject, blood glucose concentration shows a slight upward shift and returns to normal within 2-3 hours. In a diabetic subject, blood glucose concentration is generally higher than normal after fasting, rises more after the subject drinks the glucose solution, and may take several hours to return to normal. An OGTT of greater than or equal to 140 mg/dl and less than 200 mg/dl indicates that a subject has pre-diabetes. An OGTT of greater than or equal to 200 mg/dl indicates that a subject has frank diabetes, and an OGTT of less than 140 mg/dl indicates that a subject is normal (healthy) and does not have pre-diabetes or diabetes.

Overweight: An individual who weighs more than their ideal body weight. An overweight individual can be obese, but is not necessarily obese. In one embodiment, an overweight human individual is any individual who desires to decrease their weight. In another embodiment, an overweight human individual is an individual with a BMI of 25.0 kg/m² to 29.9 kg/m². Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred. The terms "polypeptide" or "protein" or "peptide" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" or "protein" or "peptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. It should be noted that the term "polypeptide" or "protein" includes naturally occurring modified forms of the proteins, such as glycosylated forms.

Pre-diabetes: A condition identified in a subject by impaired glucose tolerance, alone or in combination with impaired fasting glucose regulation. An oral glucose tolerance test (OGTT) can be used to determine if a subject has impaired glucose tolerance. An OGTT of greater than or equal to 140 mg/dl and less than 200 mg/dl indicates that a subject has pre-diabetes. An OGTT of greater than or equal to 200 mg/dl indicates that a subject has frank diabetes, and an OGTT of less than 140 mg/dl indicates that a subject is normal (healthy) and does not have pre-diabetes or diabetes. Generally, a fasting plasma glucose test (FPG) can also be used to identify a subject as pre-diabetic. A FPG of greater than 100 mg/dl and less than 126 mg/dl indicates that a subject has pre-diabetes. A FPG greater than or equal to 126 mg/dl indicates that a subject has frank diabetes, and an FPG of equal to or less than 100 mg/dl indicates that subject is normal (healthy) and does not have pre-diabetes or diabetes.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic (including a prophylactic effect) when properly administered to a subject. The pharmaceutically acceptable salts of the compounds of this invention include, but are not limited to, those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. This term refers to pharmaceutical agents, pharmaceutical compositions, and drugs acceptable for both human and veterinary uses.

Proteome: A significant portion of proteins in a biological sample at a given time. The concept of proteome is fundamentally different from the genome. While the genome is virtually static, the proteome continually changes in response to internal and external events. A "proteomic profile" is a representation of the expression pattern of a plurality of proteins in a biological sample, such as saliva, at a given time. The proteomic profile can, for example, be represented as a mass spectrum, but other representations based on any physicochemical or biochemical properties of the proteins are also included. Thus the proteomic profile may, for example, be based on differences in the electrophoretic properties of proteins, as determined by two-dimensional gel electrophoresis, e.g. by 2-D PAGE, and can be represented, e.g. as a plurality of spots in a two-dimensional electrophoresis gel. Differential expression profiles may have important diagnostic value, even in the absence of specifically identified proteins. Single protein spots can then be detected, for example, by immunoblotting, multiple spots or proteins using protein microarrays. The proteomic profile typically represents or contains information that could range from a few peaks to a complex profile representing 50 or more peaks. Thus, for example, the proteomic profile may contain or represent at least 2, or at least 5 or at least 10 or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50 proteins. A "unique expression signature" is a unique feature or motif within the proteomic profile of a biological sample (such as a reference sample) that differs from the proteomic profile of a corresponding normal biological sample (obtained from the same type of biological fluid) in a statistically significant manner. Subject: A term that includes both human and veterinary individuals, for example mammals, such as humans. Therapeutic agent: A substance that demonstrates some therapeutic effect by restoring or maintaining health, such as by alleviating the symptoms associated with a disease or physiological disorder, or delaying (including preventing) progression or onset of a disease. In some instances, the therapeutic agent is a chemical or pharmaceutical agent, or a prodrug. A therapeutic agent may be an antidiabetic agent, which includes an antihyperglycemic agent, such as an agent capable of regulating insulin levels or glucose tolerance. As one non- limiting example, the antidiabetic agent is a biguanide antidiabetic agent suitable for administration to humans. Another non-limiting example of a therapeutic agent is an incretin.

A "therapeutically effective amount" or "therapeutically effective dose" is that amount or dose sufficient to inhibit or prevent onset or advancement, to treat outward symptoms, or to cause regression, of a disease. The therapeutically effective amount or dose also can be considered as that amount or dose capable of relieving symptoms caused by the disease. Thus, a therapeutically effective amount or dose of an antidiabetic agent is that amount or dose sufficient to achieve a stated therapeutic effect. As one specific, non-limiting example, a therapeutically effective amount of an antidiabetic agent is an amount that reduces the signs of, symptoms of, or laboratory findings associated with pre-diabetes; delays the progression of pre- diabetes to diabetes; or lowers FPG or OGTT plasma glucose levels.

Transthyretin: A serum and cerebrospinal fluid carrier of the thyroid hormone tyroxine (T4), originally called prealbumin. TTR is a 55 kDa homotetramer with a dimer of dimers configuration that is synthesized in the liver, choroid plexus and retinal pigment epithelium. Each monomer is a 127 residue polypeptide rich in beta sheet structure. Association of two monomers forms an extended beta sandwich. Further association of another identical set of monomers produces the homotetrameric structure. An exemplary amino acid sequence is set forth as GENB ANK® Accession No. Q549C7, incorporated by reference herein. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Methods for Detecting Diabetes and Pre-Diabetes and for Monitoring the Efficacy of a Therapeutic Regimen

Methods are disclosed herein that are of use to determine if a subject has a diabetic condition, including pre-diabetes or diabetes, or to monitor the efficacy of therapy. The methods can be used to determine if a subject has type 1 or type 2 diabetes, or to monitor the efficacy of a therapy for either type ldiabetes or type 2 diabetes. These methods utilize a biological fluid, such as, but not limited to saliva, for the detection of biomarkers. These biomarkers can be proteins, including any naturally occurring forms of the proteins, such as but not limited to glycosylated forms. In some embodiments the subject is obese or overweight. The method can also include measuring blood hemoglobin AlC as an adjunct to the detection of other biomarkers.

In some embodiments, the methods disclosed herein are used to identify a subject as having pre-diabetes. In some embodiments, a fasting plasma glucose (FPG) test or an oral glucose tolerance test (OGTT) is also performed. In some embodiments, the methods can be used to confirm that a subject has pre-diabetes, such as a subject who has a FPG of about 100 mg/dl to about 126 mg/dl and/or an OGTT of about 140 to about 200 mg/dl. The present methods can also be used to detect pre-diabetes in a subject who is at risk for developing diabetes, such as a in an obese or overweight subject. Thus, in some embodiments, the subject of interest has a body mass index (BMI) greater than or equal to about 30 kg/m², has a family history of diabetes, or who has had gestational diabetes. The methods can be used to detect pre-diabetes in a subject who has not had a FPG or an OGTT, or a subject who has a FPG of about 90 mg/dl to about 110 mg/dl, such as about 100 mg/dl, or an OGTT of about 135 mg/dl to about 145 mg/dl, such as about 140 mg/dl.

Alternatively (or in addition) the method is used in subjects with an elevated serum hemoglobin AlC level, such as greater than about 6%. However, in other embodiments, an FPG or an OGTT is not performed on the subject. These methods can be performed over time, to monitor the progression of diabetes in a subject, or to assess for the development of diabetes from a pre-diabetic condition.

Methods are also provided for determining the efficacy of a therapy, including lifestyle modifications, for the treatment of diabetes or pre-diabetes, or preventing the development of diabetes or pre-diabetes. In one embodiment, the therapeutic regimen includes the use of at least one of metformin, insulin, incretin, lifestyle modification or dipetidyl peptidase-4 (DPP-4) inhibitors. In one embodiment, methods are provided for managing a therapeutic intervention over time. For example, the method can be used to determine whether lifestyle modifications alone are sufficient treatment, or whether pharmaceutical intervention needs to be added to a therapeutic plan. Methods are disclosed herein that include testing a biological sample, such as a saliva sample, obtained from the subject. In one example, the biological sample is a biological fluid, such as saliva. However, other biological fluids are also of use, such as blood, GCF, serum, amniotic fluid, urine or tears. The methods include detecting, or determining the abundance (amount) of one or more proteins from Table 1 below. Table 1. Exemplary proteins of a pre-diabetes or diabetes proteomic profile Swiss Prot # Name SEQ ID NO:

Q9NP55 Protein Plunc 1

P07998 Pancreatic ribonuclease 2

P19827 Inter-α-trypsin inhibitor heavy chain Hl 3

Q14624 Inter-α-trypsin inhibitor heavy chain H4 4

P80303 Nucleobindin-2 5

P26038 Moesin 6

P62258 14-3-3 epsilon 7

PO 1040 Cystatin A 8

P12429 Annexin A3 9

P31151 Protein S100-A7 10

P00558 Phosphoglycerate kinase 1 11

P04083 Annexin Al 12

P67936-2 Isoform2 of P67936 Tropomyosin α-4 13

043240 Kallikrein-10 14

P15924 Desmoplakin 15

P30043 Flavin reductase 16

P28676 Grancalcin 17

P27824 Calnexin 18

For each of these proteins, an exemplary GENB ANK® Accession number is listed. The amino acid sequences, set forth in GENB ANK® on March 10, 2008, are incorporated by reference herein. The methods can include detecting at least one, two, three, four, five, at least ten, or at least fifteen of these proteins. In some examples, the methods include determining a proteomic profile. In other examples, the methods include detecting a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins, including any of the proteins set forth in the tables herein. In some examples, the proteomic profile includes at least two of the proteins of Table 1 (such as protein plunc and pancreatic ribonuclease; inter-α-trypsin inhibitor heavy chain Hl and inter-α-trypsin inhibitor heavy chain H4; nucleobindin- 2 and moesin; and so on). In other examples, the proteomic profile includes at least three of the proteins of Table 1 (for example, protein plunc, pancreatic ribonuclease and inter-α-trypsin inhibitor heavy chain Hl; inter-α-trypsin inhibitor heavy chain H4, nucleobindin-2, and moesin; 14-3-3 epsilon, cystatin A, and annexin A3; and so on). In further examples, the proteomic profile includes at least four of the proteins of Table 1 (such as protein plunc, pancreatic ribonuclease inter-α-trypsin inhibitor heavy chain Hl, and inter-α-trypsin inhibitor heavy chain H4; nucleobindin-2, moesin, 14-3-3 epsilon, and cystatin A; annexin A3, protein S100-A7, phosphoglycerate kinase 1, and annexin Al; and so on). It is understood that any combination of any number of the proteins of Table 1 are contemplated herein.

In some embodiments, the method includes detecting an increase, such as a statistically significant increase, such as at least a 2, 3, 4, 5, 6 or 7 fold increase, in the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, and/or moesin as compared to a reference sample. In additional embodiments, the method includes detecting a decrease, such as a significantly significant increase, such as at least a 2, 3, 4, 5, 6 or 7 fold decrease in the amount of 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and/or calnexin as compared to a reference sample.

In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α- trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins with a proteomic profile from of a reference sample. In one embodiment, the method determines if the subject has pre-diabetes or diabetes. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined not to have pre-diabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample the subject is determined to have pre-diabetes or diabetes, respectively.

In another embodiment, if the reference sample is a sample from a subject with pre-diabetes or diabetes, and its proteomic profile shares at least one unique expression signature characteristic with the reference sample, then the subject is determined to have pre-diabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to have pre-diabetes or diabetes, respectively. Hence, the proteomic profile provides an additional diagnostic criterion for these disorders.

In another embodiment, the method is a method to determine if a therapy is effective for the treatment of the subject by detecting the presence of at least one protein from Table 1. The methods can include detecting at least one, two, three, four, five, at least ten, at least fifteen of these proteins. In some examples, the methods include determining a proteomic profile. In other examples, the methods include detecting a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins. The method can be performed multiple times over a specified time period, such as days, weeks, months or years. In several examples, the therapy includes treatment with metformin, dipeptidyl peptidase-4 inhibitors, or an incretin. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined to have an effective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample to have an ineffective therapy. If the reference sample is a sample from a subject with pre-diabetes or diabetes, and proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have an ineffective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined to have an effective therapy. In several examples, the therapy includes treatment with metformin, dipeptidyl peptidase-4 inhibitors, or an incretin. Changes in the profile can also represent the progression (or regression) of the disease process.

In some embodiments, the method also includes detecting, or determining the abundance (amount) of one or more proteins from Table 2 below.

Table 2. Exemplary proteins of a pre-diabetes or diabetes proteomic profile

Swiss Prot # Name SEO ID NO:

Q6FHH3 Uteroglobin 19

P23280 Carbonic anhydrase 6 20

P14618 Pyruvate kinase isozymes M1/M2 21

PO 1009 Alpha- 1 -antitrypsin 22

P22894 Neutrophil collagenase 23

PO 1023 Alpha 2-macroglobulin 24

PO 1034 Cystatin C 25

P00491 Purine nucleoside phosphorylase 26

P30838 Aldehyde dehydrogenase 27

Q01469 Fatty acid binding protein, epidermal 28

Q06830 Peroxiredoxin -1, -2, + -6 29

Q5TC18 Lamin A/C 30

Q13787 Apolipoprotein B-100 31

P07355 Annexin A2 32

P00915 Carbonic anhydrase 1 33

P00918 Carbonic anhydrase 2 34

P02763 Alpha 1 acid glycoprotein 35

P80188 Lipocalin 2 36

For each of these proteins, an exemplary GENB ANK® Accession number is listed. The amino acid sequences, set forth in GENB ANK® on March 10, 2008, are incorporated by reference herein. The methods can include detecting at least one, two, three, four, five, at least ten, at least fifteen of these proteins. In some examples, the methods include determining a proteomic profile. In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1 -antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-IOO, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin 2 as compared to the proteomic profile of a reference sample. In some examples, the proteomic profile includes all of these proteins. In other examples, the methods include detecting a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins.

In some examples, the proteomic profile includes at least two of the proteins of Table 2 (such as uteroglobin and carbonic anhydrase 6; pyruvate kinase isozymes M1/M2 and alpha- 1-antitrypsin; neutrophil collagenase and alpha 2-macroglobulin; cystatin C and purine nucleoside phosphorylase; aldehyde dehydrogenase and fatty acid binding protein, epidermal; peroxiredoxin-1, -2, -6 and lamin A/C; apolipoprotein B-100 and annexin A2; carbonic anhydrase 1 and carbonic anhydrase 2; or alpha 1 acid glycoprotein and lipocalin 2). In other examples, the proteomic profile includes at least three of the proteins of Table 2 (for example, uteroglobin, carbonic anhydrase 6, and pyruvate kinase isozymes M1/M2; alpha- 1-antitrypsin, neutrophil collagenase, and alpha 2-macroglobulin; cystatin C, purine nucleoside phosphorylase, and aldehyde dehydrogenase; fatty acid binding protein, epidermal, peroxiredoxin-1, -2, -6, and lamin A/C; apolipoprotein B-100, annexin A2, and carbonic anhydrase 1; or carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin 2). In further examples, the proteomic profile includes at least four of the proteins of Table 2 (such as uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, and alpha- 1-antitrypsin; neutrophil collagenase, alpha 2- macroglobulin, cystatin C, and purine nucleoside phosphorylase; aldehyde dehydrogenase; fatty acid binding protein, epidermal, peroxiredoxin-1, -2, -6, and lamin A/C; apolipoprotein B-100, annexin A2, carbonic anhydrase 1 and carbonic anhydrase 2 or carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin T). In additional examples, the proteomic profile includes at least five of the proteins of Table 2 (for example, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1-antitrypsin, and neutrophil collagenase; alpha 2-macroglobulin, cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, and fatty acid binding protein, epidermal; peroxiredoxin-1, -2, -6, lamin A/C, apolipoprotein B-IOO, annexin A2, and carbonic anhydrase 1; or annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin T). In further examples, the proteomic profile includes at least six of the proteins of Table 2 (for example, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1-antitrypsin, neutrophil collagenase, and alpha 2- macroglobulin; cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid binding protein, epidermal, peroxiredoxin-1, -2, -6, and lamin A/C; apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2 alpha 1 acid glycoprotein, and lipocalin T) or at least nine of the proteins of Table 2 (for example, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1-antitrypsin, neutrophil collagenase, alpha 2- macroglobulin, cystatin C, purine nucleoside phosphorylase, and aldehyde dehydrogenase; or fatty acid binding protein, epidermal, peroxiredoxin-1, -2, -6, lamin A/C; apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2 alpha 1 acid glycoprotein, and lipocalin T). It is understood that any combination of any number of the proteins of Table 2 are contemplated herein.

In some embodiments, the method includes detecting an increase, such as a statistically significant increase, such as at least a 1.5, 2, 3, 4, or 5 fold increase in the amount of uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1-antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, alpha 1 acid glycoprotein and/or lipocalin 2 as compared to a reference sample. In some embodiments, the method includes detecting an decrease, such as a statistically significant decrease, such as at least a 2, 3, 4, or 5 fold decrease in the amount of purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-100, annexin A2, carbonic anhydrase 1, and/or carbonic anhydrase 2 as compared to a reference sample.

In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha-1- antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-IOO, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin 2. Statistical methods for determining if the abundance of a protein of interest is increased relative to a control are well known in the art, and are described below.

In one embodiment, the method determines if the subject has pre-diabetes or diabetes comprising detecting the presence of at least one protein listed in Table 2. In other examples, the methods include detecting a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins and comparing the profile to a reference sample. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined not to have pre- diabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample the subject is determined to have pre-diabetes or diabetes, respectively. If the reference sample is a sample from a subject with pre-diabetes or diabetes, and the proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have prediabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to have pre-diabetes or diabetes, respectively.

In another embodiment, the method determines if a therapy is effective for the treatment of the subject. Thus, the method can be performed multiple times over a specified time period, such as days, weeks, months or years. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined to have an effective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample to have an ineffective therapy. In another embodiment, if the reference sample is a sample from a subject with pre-diabetes or diabetes, and proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have an ineffective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined to have an effective therapy. In several examples, the therapy includes treatment with metformin, dipeptidyl peptidase-4 inhibitors, or an incretin.

In further embodiments, the method includes detecting the presence or abundance (amount) of one or more of the proteins of Table 3, below.

Table 3. Exemplary proteins of a pre-diabetes or diabetes proteomic profile

Swiss Prot # Name SEO ID NO:

Q86U62 Proteasome subunit 37

O60218 Aldo-keto reductase family 1 member BlO 38 Q9UBR2 Cathepsin Z 39

Q13231-3 Chitotriosidase isoform 2, 3, + 4 40

060235 Transmembrane protease, serine 1 ID 41

Q549C7 Transthyretin 42

P06737 Glycogen phosphorylase, liver form 43 P22626 Heterogeneous nuclear RNPs A2/B 1 44

P30740 Leukocyte elastase inhibitor 45

Pl 3671 Complement component C6 46

Q4VAX6 Serpin peptidase inhibitor 47

Q96RM1 Small proline-rich protein 2F 48 Q9NZT1 Calmodulin-like protein 5 49

Q09666 Neuroblast differentiation AHNAK 50 Q4VB24 Histone cluster 1, HIe 51

Q9UKR3 Kallikrein-13 52

P36222 Chi tinase-3 -like protein 1 53

A2RTY6 Inter- alpha (Globulin) inhibitor H2 54

Q04917 14-3-3 protein eta 55

P23528 Cofilin-1 56

Q5VY30 Retinol binding protein 4, plasma 57

Q7M4Q5 Basic proline-rich peptide lB-8a 58

P60953-2 Isoform 2 of P60953 cdc 42 homolog 59

015511 Actin-related protein 2/3 complex subunit 5 60

095274 Ly6/PLAUR domain-containing protein 3 61

P61160 Actin-like protein 2 62

Q7Z3Y5 Rearranged VKA17 V gene segment 63

P80723 Brain acid soluble protein 1 64

Q8NBJ4 Golgi phosphoprotein 2 65

Q9NUQ9 Protein FAM49B (Ll) 66

P39687 Acidic leucine-rich nuclear phosphoprotein 32 67

For each of these proteins, an exemplary GENB ANK® Accession number is listed. The amino acid sequences, set forth in GENBANK® on March 10, 2008, are incorporated by reference herein. The methods can include detecting at least one, two, three, four, five, at least ten, fifteen, twenty, twenty-five, thirty, or all of these proteins. In some examples, the methods include determining a proteomic profile. In additional examples, the methods include detecting all of these proteins, such as a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of these proteins. In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of proteasome subunit, aldo-keto reductase family 1 member BlO, cathepsin Z, chitotriosidase isoform 2, 3, + 4, transmembrane protease, serine HD, transthyretin, glycogen phosphorylase, heterogeneous nuclear RNPs A2/B1, leukocyte elastase inhibitor, small proline-rich protein 2F, calmodulin-like protein 5, neuroblast differentiation AHNAK, histone cluster 1, HIe, kallikrein-13, chitinase- 3-like protein 1, inter-alpha (Globulin) inhibitor H2, 14-3-3 protein eta, cofilin-1, retinol binding protein 4, plasma, basic proline-rich peptide lB-8a, isoform 2 of P60953 cdc 42 homolog, actin-related protein 2/3 complex subunit 5, Iy6/PLAUR domain-containing protein 3, actin-like protein 2, Rearranged VKA17 V gene segment, brain acid soluble protein 1, golgi phosphoprotein 2, protein FAM49B (Ll), and acidic leucine-rich nuclear phosphoprotein 32 as compared to the proteomic profile of a reference sample.

In some examples, the proteomic profile includes at least two of the proteins of Table 3 (such as proteasome subunit and aldo-keto reductase family 1 member BlO; cathepsin Z and chitotriosidase isoform 2, 3, +4; transmembrane protease, serine 1 ID and transthyretin; and so on). In other examples, the proteomic profile includes at least three of the proteins of Table 3 (for example, proteasome subunit, aldo-keto reductase family 1 member BlO, and cathepsin Z; chitotriosidase isoform 2, 3, +4; transmembrane protease, serine HD, and transthyretin; glycogen phosphorylase, liver form, heterogeneous nuclear RNPs A2/B1, and leukocyte elastase inhibitor; and so on). In further examples, the proteomic profile includes at least four of the proteins of Table 3 (such as proteasome subunit, aldo-keto reductase family 1 member BlO, cathepsin Z, and chitotriosidase isoform 2, 3, +4; transmembrane protease, serine HD, transthyretin, glycogen phosphorylase, liver form, and heterogeneous nuclear RNPs A2/B1; leukocyte elastase inhibitor, complement component C6, serpin peptidase inhibitor, and small proline-rich protein 2F; and so on). It is understood that any combination of any number of the proteins of Table 3 are contemplated herein. In one embodiment, the method is a method to determine if the subject has pre-diabetes or diabetes comprising detecting at least one protein listed in Table 3. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined not to have pre-diabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample the subject is determined to have pre- diabetes or diabetes, respectively. If the reference sample is a sample from a subject with pre-diabetes or diabetes, and proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have pre-diabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to have pre-diabetes or diabetes, respectively.

In another embodiment, the method is a method to determine if a therapy is effective for the treatment of the subject. Thus, the method can be performed multiple times over a specified time period, such as days, weeks, months or years. If the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined to have an effective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample to have an ineffective therapy. If the reference sample is a sample from a subject with pre-diabetes or diabetes, and proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have an ineffective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined to have an effective therapy. In several examples, the therapy includes treatment with metformin, dipeptidyl peptidase-4 inhibitors, or an incretin. Methods for monitoring the efficacy of therapeutic agents are described below.

Monitoring

The diagnostic methods of the present invention are valuable tools for practicing physicians to make quick treatment decisions for diabetic conditions, including both pre-diabetes and diabetes. These treatment decisions can include the administration of an anti-diabetic agent and decisions to monitor a subject for onset and/or advancement of diabetes. The treatment decisions can also include lifestyle monitoring. The method disclosed herein can also be used to monitor the effectiveness of a therapy. Following the measurement of the expression levels of one or more of the proteins identified herein, the assay results, findings, diagnoses, predictions and/or treatment recommendations are typically recorded and communicated to technicians, physicians and/or patients, for example. In certain embodiments, computers will be used to communicate such information to interested parties, such as, patients and/or the attending physicians. Based on the measurement, the therapy administered to a subject can be modified.

In one embodiment, a diagnosis, prediction and/or treatment recommendation based on the expression level in a test subject of one or more of the biomarkers herein is communicated to the subject as soon as possible after the assay is completed and the diagnosis and/or prediction is generated. The results and/or related information may be communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, such as by providing a written report, electronic forms of communication, such as email, or telephone.

Communication may be facilitated by use of a computer, such as in case of email communications. In certain embodiments, the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761; however, the present invention is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the invention, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as being pre-diabetic or diabetic results in the physician treating the subject, such as prescribing an anti- hyperglycemic or an anti-diabetic agent to inhibit or delay the onset or progression of type II diabetes. In additional embodiment, the dose or dosing regimen is modified based on the information obtained using the methods disclosed herein. In some embodiments, the anti-diabetic agent contains a biguanide of the formula:

N-C — NH-C-NH₂

^R2 N N

I I

R₃ R₄

Formula 1 wherein Ri and R₂ are independently selected from alkyl, lower alkyl, alkenyl, lower alkenyl, cycloalkyl, aryl, or an arylalkyl of the formula:

wherein X is hydrogen or halogen and n = 0, 1 or 2; R₃ and R₄ are independently selected from hydrogen, alkyl, lower alkyl, alkenyl, lower alkenyl, cycloalkyl, alkoxy, lower alkoxy, alkoxyalkyl; and pharmaceutically acceptable salts thereof. In particular embodiments, the biguanide antidiabetic agent is metformin. Metformin is manufactured by Lyonnaise Industrielle Pharmaceutique SA (Lyons, France), also known by its acronym LIPHA SA, and commercially distributed in the United States as a hydrochloride salt by the Bristol-Myers Squibb Company (Princeton, New Jersey) as GLUCOPHAGE^® XR. Additionally, Bristol-Myers Squibb distributes a pharmaceutical having a combination of metformin and glyburide as GLUCOVANCE^®.

Anti-diabetic agents other than biguanides can also be administered to the identified subject. For example, in alternative embodiments, the anti-diabetic agent is a thiazolidinedione, such as troglitazone. In some examples, the anti-diabetic agent is an incretin or dipeptidyl peptidase-4 inhibitor, but the anti-diabetic agent can be any agent of interest. A therapeutically effective amount of an anti-diabetic agent may be administered in a single dose, or in several doses, for example daily, during a course of treatment. The course of treatment may last for any length of time, such as a day or several days, a week or several weeks, a month or several months, or a year or several years, so long as the therapeutic effect is observed, such as inhibiting the onset of type II diabetes in a subject diagnosed with pre-diabetes, or inducing a subject diagnosed with type 2 diabetes or pre-diabetes to a normal glucose tolerance. The subject can be monitored while undergoing treatment using the methods described herein in order to assess the efficacy of the treatment protocol. In this manner, the length of time or the amount give to the subject can be modified based on the results obtained using the methods disclosed herein.

The therapeutically effective amount will depend on the anti-diabetic agent being used, the characteristics of the subject being treated (such as age, BMI, physiological condition, etc.), the severity and type of the affliction, and the manner of administration of the agent. The therapeutically effective dose can be determined by various methods, including generating an empirical dose-response curve, predicting potency and efficacy by using quantitative structure activity relationships (QSAR) methods or molecular modeling, and other methods used in the pharmaceutical sciences. In certain, non-limiting examples, the therapeutically effective amount of metformin (or a related biguanide analog or homolog) is at least about 1000 mg per day, such as at least about 1500 mg per day, or even at least about 1700 mg per day. In certain other, non-limiting examples, the total amount of metformin is divided into smaller doses, such as two or three doses per day, for example 850 mg twice a day (b.i.d.) or 500 mg three times a day (t.i.d.). In alternative, non-limiting examples, the total amount of metformin is about 500 mg or less per day. The subject can be monitored at different doses of an agent using the assays described herein, in order to determine a therapeutically effective amount for the subject of interest.

For administration to animals, purified therapeutically active agents are generally combined with a pharmaceutically acceptable carrier. Pharmaceutical preparations may contain only one type of anti-diabetic agent, or may be composed of a combination of several types of anti-diabetic agents, such as a combination of two or more anti-diabetic agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Anti-diabetic agents may be administered by any means that achieve their intended purpose. For example, the anti-diabetic agents may be administered to a subject through systemic administration, such as intravenous or intraperitoneal administration; intralesionally; by suppository; or orally.

The anti-diabetic agent can be administered alone or in combination with another anti-diabetic agent. In certain embodiments, the anti-diabetic agent is administered in the absence of administering any other anti-diabetic agent.

Other measures may be taken to inhibit or delay the onset of type II diabetes in subjects at a heightened risk of developing the disease. For example, in some embodiments, a subject may be instructed, trained, or induced to adopt anti-diabetic lifestyle modifications. For example, the subject can be counseled to reduce caloric intake or to exercise. The methods disclosed herein can be used to monitor the effectiveness of these alternative measures, to determine if pharmaceutical intervention is warranted for a subject of interest.

Methods for Determining Risk Methods are provided herein to determine if a subject is at risk for developing a diabetic condition, such as pre-diabetes or diabetes. These methods utilize a biological fluid, such as, but not limited to saliva, for the detection of biomarkers. These biomarkers can be proteins, including any naturally occurring forms of the proteins, such as but not limited to glycosylated forms. The method can detect the risk for developing type 1 or type 2 diabetes. In some embodiments the subject is obese or overweight. These biomarkers can be proteins, including any naturally occurring forms of the proteins, such as but not limited to glycosylated forms. In some embodiments the subject is obese or overweight. The method can also include measuring blood hemoglobin AlC as an adjunct to the detection of other biomarkers. In some embodiments, the methods disclosed herein are used to identify a subject as having pre-diabetes. In some embodiments, an FPG or an OGTT is not performed on the subject. These methods can be performed over time, to determine if the subject is at increased or decreased risk for developing pre-diabetes or diabetes. For example, the methods can be performed after an environmental exposure to determine if the exposure increases the subject's risk for developing diabetes. The methods can also be performed over time, to determine if a subject's risk for developing diabetes or pre-diabetes has increased or decreased over time. In some examples, the method is used in a subject with an elevated serum AlC level, such as greater than about 5%. Methods are disclosed herein that include testing in a biological sample, such as a saliva sample, obtained from the subject. In one example, the biological sample is a biological fluid, such as saliva. However, other biological fluids are also of use, such as blood, GCF, serum, amniotic fluid, urine or tears.

The methods include detecting, or determining the abundance (amount) of one or more proteins from Table 1 above. The methods can include detecting at least one, two, three, four, five, at least ten, or at least fifteen of these proteins. In some examples, the methods include determining a proteomic profile. In other examples, the methods include detecting all of these proteins, such as a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins. In some embodiments, the method includes detecting an increase, such as a significantly significant increase, such as at least a 2, 3, 4, 5, 6 or 7 fold increase in the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, and/or moesin as compared to a reference sample. In additional embodiments, the method includes detecting a decrease, such as a statistically significant decrease, such as at least a 2, 3, 4, 5, 6 or 7 fold decrease in the amount of 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and/or calnexin as compared to a reference sample, such as a sample from a subject that does not have diabetes and/or is known not to be at risk for developing diabetes.

In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α- trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins with a proteomic profile from of a reference sample. In one embodiment, if the reference sample is a normal sample, or a sample from a subject known not to be at risk for developing diabetes or pre-diabetes, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined not to be at risk for prediabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the reference sample the subject is determined to be at risk for pre-diabetes or diabetes, respectively.

If the reference sample is a sample from a subject with pre-diabetes or diabetes, or a subject known to be at risk for developing pre-diabetes or diabetes, and its proteomic profile shares at least one unique expression signature characteristic with the reference sample, then the subject is determined to be at risk for pre-diabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to be at risk for pre-diabetes or diabetes, respectively. The proteomic profile can provide an additional criterion for the identification of a subject at risk for a diabetic condition.

In some embodiments, the method also includes detecting, or determining the abundance (amount) of one or more proteins from Table 2 above. The methods can include detecting at least one, two, three, four, five, at least ten, at least fifteen or all of these proteins. In some examples, the methods include determining a proteomic profile. In other examples, the methods include a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of these proteins (for example, one or more of the combinations described above). In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, alpha- 1 -antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin 2. In some embodiments, the method includes detecting an increase, such as a statistically significant increase, such as at least a 1.5, 2, 3, 4, or 5 fold increase, in the amount of uteroglobin, carbonic anhydrase C, pyruvate kinase isozymes M1/M2, alpha- 1 -antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, alpha 1 acid glycoprotein, and/or lipocalin 2 as compared to a reference sample. In some embodiments, the method includes detecting an decrease, such as a statistically significant decrease, such as at least a 2, 3, 4, or 5 fold decrease in the amount of purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-100, annexin A2, carbonic anhydrase 1, and/or carbonic anhydrase 2 as compared to a reference sample. In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of uteroglobin, carbonic anhydrase C, pyruvate kinase isozymes M1/M2, alpha-1- antitrypsin, neutrophil collagenase, alpha 2-macroglobulin, cystatin C, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-IOO, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, alpha 1 acid glycoprotein, and lipocalin 2.

Statistical methods for determining if the abundance of a protein of interest is increased relative to a reference sample are well known in the art, and are described below. If the reference sample is a normal sample, or a sample from a subject known not to be at risk for pre-diabetes and/or diabetes, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the reference sample the subject is determined not to be at risk for pre-diabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the reference sample the subject is determined to be at risk for pre-diabetes or diabetes, respectively. If the reference sample is a sample from a subject with pre-diabetes or diabetes, or known to be at risk for pre-diabetes and/or diabetes, and the proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to be at risk for pre-diabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to be at risk for pre-diabetes or diabetes, respectively. The expression signature can include a difference in a single or multiple proteins in comparison to a control subject.

In yet further embodiments, the method includes detecting the presence or abundance (amount) of one or more of the proteins of Table 3 above. The methods can include detecting at least one, two, three, four, five, at least ten, fifteen, twenty, twenty-five, thirty, or all of these proteins. In some examples, the methods include determining a proteomic profile. In other examples, the methods include detecting all of these proteins, such as a proteomic profile including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of these proteins. In another example, the methods include detecting all of these proteins. In one embodiment, the method includes comparing a proteomic profile of a test sample of saliva from a subject of interest comprising at least one of proteasome subunit, aldo-keto reductase family 1 member BlO, cathepsin Z, chitotriosidase isoform 2, 3, + 4, transmembrane protease, serine HD, transthyretin, glycogen phosphorylase, heterogeneous nuclear RNPs A2/B1, leukocyte elastase inhibitor, small proline-rich protein 2F, calmodulin-like protein 5, neuroblast differentiation AHNAK, histone cluster 1, HIe, kallikrein-13, chitinase-3-like protein 1, inter-alpha (Globulin) inhibitor H2, 14-3-3 protein eta, cofilin-1, retinol binding protein 4, plasma, basic proline-rich peptide lB-8a, isoform 2 of P60953 cdc 42 homolog, actin-related protein 2/3 complex subunit 5, Iy6/PLAUR domain-containing protein 3, actin-like protein 2, Rearranged VKA 17 V gene segment, brain acid soluble protein 1, golgi phosphoprotein 2, protein FAM49B (Ll), and acidic leucine-rich nuclear phosphoprotein 32. If the reference sample is a normal sample or a sample from a subject known not to be at risk for pre-diabetes and/or diabetes, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the reference sample the subject is determined not to be at risk for pre-diabetes or diabetes, respectively. However, if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the reference sample the subject is determined to be at risk for pre-diabetes or diabetes, respectively. If the reference sample is a sample from a subject with pre-diabetes or diabetes, or known to be at risk for pre-diabetes and/or diabetes, and the proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to be at risk for pre-diabetes or diabetes, respectively. If the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to be at risk for pre-diabetes or diabetes, respectively. Proteomic Identification of Proteins and Polypeptides Expressed in Biological

Fluids

Proteomic analysis of biological fluids, such as saliva or serum, can be performed using a variety of methods known in the art, and are of use in the methods disclosed herein. The biological fluid can be saliva, such as from a subject of interest, and/or a subject with diabetes, and/or a subject with pre-diabetes and/or a control subject without diabetes (or pre-diabetes). Typically, protein patterns (proteome maps) of samples from different sources, such as normal biological fluid (normal sample) and a test biological fluid (test sample), are compared to detect proteins that are up- or down-regulated in a disease, such as diabetes. These proteins can then be excised for identification and full characterization, such as by using peptide-mass fingerprinting and/or mass spectrometry and sequencing methods, or the normal and/or disease-specific proteome map can be used directly for the diagnosis of the disease of interest, or to confirm the presence or absence of the disease, such as pre-diabetes or diabetes.

In a direct comparative analysis, the reference, such as a normal (such as a sample from a non-diabetic subject) or known (such as a sample from a subject known to have diabetes or pre-diabetes) and test samples are treated exactly the same way, in order to correctly represent the relative abundance of proteins, and obtain accurate results. The required amount of total proteins depends on the analytical technique used, and can be readily determined by one skilled in the art. The proteins present in the biological samples are typically separated by two- dimensional gel electrophoresis (2-DE) according to their pi and molecular weight. The proteins are first separated by their charge using isoelectric focusing (one- dimensional gel electrophoresis). This step can, for example, be carried out using immobilized pH-gradient (IPG) strips, which are commercially available. The second dimension is a normal SDS-PAGE analysis, where the focused IPG strip is used as the sample. After 2-DE separation, proteins can be visualized with conventional dyes, like Coomassie Blue or silver staining, and imaged using known techniques and equipment, such as, for example Bio-Rad GS800 densitometer and PDQUEST™ software, both of which are commercially available. Individual spots are then cut from the gel, destained, and subjected to tryptic digestion. The peptide mixtures can be analyzed by mass spectrometry (MS). Alternatively, the peptides can be separated, for example by capillary high pressure liquid chromatography (HPLC) and can be analyzed by MS either individually, or in pools. Mass spectrometers consist of an ion source, mass analyzer, ion detector, and data acquisition unit. First, the peptides are ionized in the ion source. The ionized peptides then are separated according to their mass-to-charge ratio in the mass analyzer and the separate ions are detected. Mass spectrometry has been widely used in protein analysis, especially since the invention of matrix-assisted laser-desorption ionisation/time-of-flight (MALDI-TOF) and electrospray ionisation (ESI) methods. There are several versions of mass analyzer, including, for example, MALDI-TOF and triple or quadrupole-TOF, or ion trap mass analyzer coupled to ESI. Thus, for example, a Q-Tof-2 mass spectrometer utilizes an orthogonal time- of-flight analyzer that allows the simultaneous detection of ions across the full mass spectrum range (see, for example, Chemusevich et ah, J. Mass Spectrom. 36:849- 865, 2001, incorporated herein by reference).

If desired, the amino acid sequences of the peptide fragments and eventually the proteins from which they derived can be determined by techniques known in the art, such as certain variations of mass spectrometry, or Edman degradation. A "proteomic profile" refers to a representation of the expression pattern of a plurality of proteins in a biological sample, such as biological fluid (for example, saliva, blood or serum) at a given time. The proteomic profile can, for example, be represented as a mass spectrum, but other representations based on any physicochemical or biochemical properties of the proteins are also included. Although it is possible to identify and sequence all or some of the proteins present in the proteome of a biological fluid, this is not necessary for the diagnostic use of the proteomic profiles.

Diagnosis of a diabetes or pre-diabetes can be based on characteristic differences (unique expression signatures) between a normal proteomic profile (a profile from a non-diabetic subject, who does not have pre-diabetes or diabetes), and the proteomic profile of the same biological fluid obtained under the same experimental conditions. Diagnosis of a diabetes or pre-diabetes can be based on characteristic similarities (unique expression signatures) between a diabetic or pre- diabetic proteomic profile respectively, and the proteomic profile of the test biological fluid obtained under the same experimental conditions. The unique expression signature can be any unique feature or motif within the proteomic profile of a test or reference biological sample that differs from the proteomic profile of a corresponding normal biological sample obtained from the same type of source, in a statistically significant manner. For example, if the proteomic profile is presented in the form of a mass spectrum, the unique expression signature is typically a peak or a combination of peaks that differ, qualitatively or quantitatively, from the mass spectrum of a corresponding normal sample. Thus, the appearance of a new peak or a combination of new peaks in the mass spectrum, or any statistically significant change in the amplitude or shape of an existing peak or combination of existing peaks, or the disappearance of an existing peak, in the mass spectrum can be considered a unique expression signature.

When the proteomic profile of the test sample obtained from a subject of interest is compared with the proteomic profile of a reference sample comprising a unique expression signature characteristic of a pre-diabetic or diabetic subject, the subject of interest is diagnosed with diabetes or pre-diabetes if it shares the unique expression signature with the reference sample. For example a profile that does not have any statistically significant change in the amplitude or shape of an existing peak representing one or more of alpha- 1- antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR) from a sample from a subject with pre-diabetes or diabetes, detects pre-diabetes or diabetes in the subject, respectively. In one example, if the proteomic profile of the test sample shows a unique expression signature, such as an increase in the abundance of one, two, three or four of alpha- 1-antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR) relative to the proteomic profile of the normal biological fluid, then the subject has pre-diabetes. In a specific, non-limiting example, if the proteomic profile of a test sample shows a unique expression signature, such as an increase in the abundance of one, two, three or four of alpha- 1- antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR) relative to the proteomic profile of a biological subject with prediabetes, then the subject has diabetes. Other combinations (such as those as described above) can also be used to detect a proteomic profile from a subject of interest to diagnose whether the subject has pre-diabetes or diabetes.

Pre-diabetes or diabetes also can be diagnosed by comparing the proteomic profile of a biological fluid obtained from the subject to be diagnosed with the proteomic profile of a normal biological fluid of the same kind, obtained and treated the same manner. If the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample (from a subject without diabetes or pre-diabetes), the subject is considered not to have pre-diabetes or diabetes, respectively). If the proteomic profile of the test sample shows a unique expression signature, such as an increase in the abundance of one, two, three or four of alpha- 1- antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR) relative to the proteomic profile of the normal biological fluid, then the subject has pre-diabetes. If the proteomic profile of the test sample shows a unique expression signature, such as an increase in the abundance of one, two, three or four of alpha- 1-antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR) relative to the proteomic profile of the biological fluid from a subject with pre-diabetes, then the subject has diabetes.

Alternatively or in addition, the proteomic profile of the test sample may be compared with the proteomic profile of a reference standard, such as a previously established proteomic profile or a set of values from a subject with pre-diabetes or diabetes. In this case, the subject is diagnosed with the pathologic condition if the proteomic profile of the test sample shares at least one feature, or a combination of features representing a unique expression signature, with the proteomic profile of the reference sample from the subject with pre-diabetes or diabetes.

Statistical methods for comparing proteomic profiles are well known in the art. For example, in the case of a mass spectrum, the proteomic profile is defined by the peak amplitude values at key mass/charge (M/Z) positions along the horizontal axis of the spectrum. Accordingly, a characteristic proteomic profile can, for example, be characterized by the pattern formed by the combination of spectral amplitudes at given M/Z vales. The presence or absence of a characteristic expression signature, or the substantial identity of two profiles can be determined by matching the proteomic profile (pattern) of a test sample with the proteomic profile (pattern) of a reference or normal sample, with an appropriate algorithm. Statistical methods for analyzing proteomic patterns is disclosed, for example, in Petricoin III, et al., The Lancet 359:572-77, 2002; Issaq et al., Biochem Biophys Commun 292:587-92 (2002); Ball et al., Bioinformatics 18:395-404, 2002; and Li et al., Clinical Chemistry Journal, 48: 1296-1304, 2002.

Protein Arrays

The disclosed methods can utilize protein arrays, which can be used to detect proteins, monitor their expression levels, and investigate protein interactions and functions. The use of protein arrays enables high-throughput protein analysis, when large numbers of determinations can be performed simultaneously, using automated means. In the microarray or chip format, that was originally developed for DNA arrays, such determinations can be carried out with minimum use of materials while generating large amounts of data. Protein microarray s, in addition to their high efficiency, provide very high sensitivity. Protein arrays are formed by immobilizing proteins on a solid surface, such as glass, silicon, micro-wells, nitrocellulose, PVDF membranes, and microbeads, using a variety of covalent and non-covalent attachment chemistries well known in the art. The solid support is chemically stable before and after the coupling procedure, allow good spot morphology, display minimal nonspecific binding, should not contribute a background in detection systems, and should be compatible with different detection systems. In general, protein microarrays use the same detection methods commonly used for the reading of DNA arrays. Similarly, the same instrumentation as used for reading DNA microarrays is applicable to protein arrays. Thus, capture arrays (such as antibody arrays) can be probed with fluorescently labeled proteins from two different sources, such as normal and diabetic and/or pre-diabetic biological fluids. In this case, the readout is based on the change in the fluorescent signal as a reflection of changes in the expression level of a target protein. Alternative readouts include, without limitation, fluorescence resonance energy transfer, surface plasmon resonance, mass spectrometry, resonance light scattering, and atomic force microscopy (see Zhou H, et al., Trends Biotechnol. 19:S34-9, 2001; Zhu et al., Current Opin. Chem. Biol. 5:40-45, 2001; Wilson and Nock, Angew Chem Int Ed Engl 42:494-500, 2003); and Schweitzer and Kingsmore, Curr Opin Biotechnol 13:14-9, 2002). Biomolecule arrays are also disclosed in United States Patent No. 6,406,921, issued June 18, 2002, the entire disclosure of which is hereby incorporated by reference. In some embodiments the capture arrays include antibodies to two, three or four of alpha- 1 -antitrypsin (AlAT), cystatin C (CysC), alpha-2-macroglobulin (A2MG), and transthyretin (TTR). The capture arrays can also include antibodies that specifically bind a protein listed in Table 2. An additional, two, three, four, five, ten, twenty, thirty, forty or fifty antibodies can be included on a capture array.

Immunoassays

The methods disclosed herein can also be performed in the form of various immunoassay formats, which are well known in the art. There are two main types of immunoassays, homogeneous and heterogeneous. In homogeneous immunoassays, both the immunological reaction between an antigen and an antibody and the detection are carried out in a homogeneous reaction. Heterogeneous immunoassays include at least one separation step, which allows the differentiation of reaction products from unreacted reagents. A variety of immunoassays can be used to detect one or more of the proteins listed in Tables 1, 2 or 3. In addition, immunoassays can be used to detect any of the proteins listed in Table 5.

ELISA is a heterogeneous immunoassay, which has been widely used in laboratory practice since the early 1970s, and can be used in the methods disclosed herein. The assay can be used to detect protein antigens in various formats. In the "sandwich" format the antigen being assayed is held between two different antibodies. In this method, a solid surface is first coated with a solid phase antibody. The test sample, containing the antigen (e.g., a diagnostic protein), or a composition containing the antigen, such as a saliva sample from a subject of interest, is then added and the antigen is allowed to react with the bound antibody. Any unbound antigen is washed away. A known amount of enzyme-labeled antibody is then allowed to react with the bound antigen. Any excess unbound enzyme-linked antibody is washed away after the reaction. The substrate for the enzyme used in the assay is then added and the reaction between the substrate and the enzyme produces a color change. The amount of visual color change is a direct measurement of specific enzyme-conjugated bound antibody, and consequently the antigen present in the sample tested.

ELISA can also be used as a competitive assay. In the competitive assay format, the test specimen containing the antigen to be determined is mixed with a precise amount of enzyme-labeled antigen and both compete for binding to an anti- antigen antibody attached to a solid surface. Excess free enzyme-labeled antigen is washed off before the substrate for the enzyme is added. The amount of color intensity resulting from the enzyme-substrate interaction is a measure of the amount of antigen in the sample tested. A heterogenous immunoassay, such as an ELISA, can be used to detect any of the proteins listed in Tables 1, 2, 3 or 5.

In another example, immuno-PCR can be used to detect any of the proteins listed in Tables 1, 2, 3, or 5. Immuno-PCR is a modification of the conventional ELISA format in which the detecting antibody is labeled with a DNA label, and is applicable to the analysis of biological samples (see, e.g., U.S. Patent No. 5,665,539 and U.S. Patent Application Publication No. 2005/0239108; all herein incorporated by reference). The amplification ability of PCR provides large amounts of the DNA label which can be detected by various methods, typically gel electrophoresis with conventional staining (e.g., Sano et al, Science, 258:120-122, 1992). This method can also include the direct conjugation of the DNA label to the antibody and replacement of gel electrophoresis by using labeled primers to generate a PCR product that can be assayed by ELISA or using real time quantitative PCR. In an example of the real-time PCR method, PCR is used to amplify DNA in a sample in the presence of a nonextendable dual labeled fluorogenic hybridization probe. One fluorescent dye serves as a reporter and its emission spectra is quenched by the second fluorescent dye. The method uses the 5' nuclease activity of Taq polymerase to cleave a hybridization probe during the extension phase of PCR. The nuclease degradation of the hybridization probe releases the quenching of the reporter dye resulting in an increase in peak emission from the reporter. The reactions are monitored in real time.

Homogeneous immunoassays include, for example, the Enzyme Multiplied Immunoassay Technique (EMIT), which typically includes a biological sample comprising the biomarkers to be measured, enzyme-labeled molecules of the biomarkers to be measured, specific antibody or antibodies binding the biomarkers to be measured, and a specific enzyme chromogenic substrate. In a typical EMIT, excess of specific antibodies is added to a biological sample. If the biological sample contains the proteins to be detected, such proteins bind to the antibodies. A measured amount of the corresponding enzyme-labeled proteins is then added to the mixture. Antibody binding sites not occupied by molecules of the protein in the sample are occupied with molecules of the added enzyme-labeled protein. As a result, enzyme activity is reduced because only free enzyme-labeled protein can act on the substrate. The amount of substrate converted from a colorless to a colored form determines the amount of free enzyme left in the mixture. A high concentration of the protein to be detected in the sample causes higher absorbance readings. Less protein in the sample results in less enzyme activity and consequently lower absorbance readings. Inactivation of the enzyme label when the antigen-enzyme complex is antibody-bound makes the EMIT a useful system, enabling the test to be performed without a separation of bound from unbound compounds as is necessary with other immunoassay methods. A homogenous immunoassay, such as an EMIT, can be used to detect any of the proteins listed in Tables 1, 2, 3 or 5.

Immunoassay kits are also disclosed herein. These kits include, in separate containers (a) monoclonal antibodies having binding specificity for the polypeptides used in the diagnosis of a pre-diabetes or diabetes; and (b) and anti-antibody immunoglobulins. This immunoassay kit may be utilized for the practice of the various methods provided herein. The monoclonal antibodies and the anti-antibody immunoglobulins can be provided in an amount of about 0.001 mg to 100 grams, and more preferably about 0.01 mg to 1 gram. The anti-antibody immunoglobulin may be a polyclonal immunoglobulin, protein A or protein G or functional fragments thereof, which may be labeled prior to use by methods known in the art. In several embodiments, the immunoassay kit includes two, three or four of: antibodies that specifically bind a protein listed in Table 1. The immunoassay kit can also include one or more antibodies that specifically bind a protein listed in Table 2. The immunoassay kit can also include one or more antibodies that specifically bind a protein listed in Table 3. In one example, the immunoassay kit includes antibodies that specifically bind one, two or three of: antibodies that specifically bind different proteins listed in Table 1, antibodies that specifically bind a protein listed in Table 2, and antibodies that specifically bind a protein listed in Table 3. Thus, the kits can be used to detect two or more different proteins listed in Tables 1, 2 and/or 3.

Capture Device Methods

The disclosed methods can be carried out using a sample capture device, such as a lateral flow device (for example a lateral flow test strip) that allows detection of one or more proteins, such as those described herein.

Point-of-use analytical tests have been developed for the routine identification or monitoring of health-related conditions (such as pregnancy, cancer, endocrine disorders, infectious diseases or drug abuse) using a variety of biological samples (such as urine, serum, plasma, blood, saliva). Some of the point-of-use assays are based on highly specific interactions between specific binding pairs, such as antigen/antibody, hapten/antibody, lectin/carbohydrate, apoprotein/cofactor and biotin/(strept)avidin. The assays are often performed with test strips in which a specific binding pair member is attached to a mobilizable material (such as a metal sol or beads made of latex or glass) or an immobile substrate (such as glass fibers, cellulose strips or nitrocellulose membranes). Particular examples of some of these assays are shown in U.S. Patent Nos. 4,703,017; 4,743,560; and 5,073,484 (incorporated herein by reference). The test strips include a flow path from an upstream sample application area to a test site. For example, the flow path can be from a sample application area through a mobilization zone to a capture zone. The mobilization zone may contain a mobilizable marker that interacts with an analyte or analyte analog, and the capture zone contains a reagent that binds the analyte or analyte analog to detect the presence of an analyte in the sample.

Examples of migration assay devices, which usually incorporate within them reagents that have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances are found, for example, in U.S. Patent No. 4,770,853; WO 88/08534; and EP-A 0 299 428

(incorporated herein by reference). There are a number of commercially available lateral-flow type tests and patents disclosing methods for the detection of large analytes (MW greater than 1,000 Daltons) as the analyte flows through multiple zones on a test strip. Examples are found in U.S. Patent No. 5,229,073 (measuring plasma lipoprotein levels), and U.S. Patent Nos. 5,591,645; 4,168,146; 4,366,241; 4,855,240; 4,861,711; 5,120,643; European Patent No. 0296724; WO 97/06439; WO 98/36278; and WO 08/030546 (each of which are herein incorporated by reference). Multiple zone lateral flow test strips are disclosed in U.S. Patent No. 5,451,504, U.S. Patent No. 5,451,507, and U.S. Patent No. 5,798,273 (incorporated by reference herein). U.S. Patent No. 6,656,744 (incorporated by reference) discloses a lateral flow test strip in which a label binds to an antibody through a streptavidin-biotin interaction.

In particular examples, the methods disclosed herein include application of a biological sample (such as saliva or urine) from a test subject to a lateral flow test device for the detection of one or more proteins (such as one or more proteins listed in Tables 1, 2, 3, and 5, for example, combinations of proteins as described above) in the sample. The lateral flow test device includes one or more antibodies (such as antibodies that bind one or more of the proteins listed in Tables 1, 2, 3, and 5) at an addressable location. In a particular example, the lateral flow test device includes antibodies that bind AlAT and AlAG. The addressable locations can be, for example, a linear array or other geometric pattern that provides diagnostic information to the user. The binding of one or more proteins in the sample to the antibodies present in the test device is detected and the presence or amount of one or more proteins in the sample of the test subject is compared to a control, wherein a change in the presence or amount of one or more proteins in the sample from the test subject as compared to the control indicates that the subject has pre-diabetes or diabetes. Flow-through devices

Flow-through type assay devices were designed, in part, to obviate the need for incubation and washing steps associated with dipstick assays. Flow- through immunoassay devices involve a capture reagent (such as one or more antibodies) bound to a porous membrane or filter to which a liquid sample is added. As the liquid flows through the membrane, target analyte (such as protein) binds to the capture reagent. The addition of sample is followed by (or made concurrent with) addition of detector reagent, such as labeled (e.g., gold-conjugated or colored latex particle-conjugated protein). Alternatively, the detector reagent may be placed on the membrane in a manner that permits the detector to mix with the sample and thereby label the analyte. The visual detection of detector reagent provides an indication of the presence of target analyte in the sample. Representative flow- through assay devices are described in U.S. Patent Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and 5,279,935; U.S. Patent Application

Publication Nos. 20030049857 and 20040241876; and WO 08/030546. Migration assay devices usually incorporate within them reagents that have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances. See, for example, U.S. Patent No. 4,770,853; PCT Publication No. WO 88/08534 and European Patent No. EP-A 0 299 428.

There are a number of commercially available lateral flow type tests and patents disclosing methods for the detection of large analytes (MW greater than 1,000 Daltons). U.S. Patent No. 5,229,073 describes a semiquantitative competitive immunoassay lateral flow method for measuring plasma lipoprotein levels. This method utilizes a plurality of capture zones or lines containing immobilized antibodies to bind both the labeled and free lipoprotein to give a semi-quantitative result. In addition, U.S. Patent No. 5,591,645 provides a chromatographic test strip with at least two portions. The first portion includes a movable tracer and the second portion includes an immobilized binder capable of binding to the analyte. Additional examples of lateral flow tests for large analytes are disclosed in the following patent documents: U.S. Patent Nos. 4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; European Patent No. 0296724; WO 97/06439; WO 98/36278; and WO 08/030546.

Devices described herein generally include a strip of absorbent material (such as a microporous membrane), which, in some instances, can be made of different substances each joined to the other in zones, which may be abutted and/or overlapped. In some examples, the absorbent strip can be fixed on a supporting non- interactive material (such as nonwoven polyester), for example, to provide increased rigidity to the strip. Zones within each strip may differentially contain the specific binding partner(s) and/or other reagents required for the detection and/or quantification of the particular analyte being tested for, for example, one or more proteins disclosed herein. Thus these zones can be viewed as functional sectors or functional regions within the test device.

In general, a fluid sample is introduced to the strip at the proximal end of the strip, for instance by dipping or spotting. A sample is collected or obtained using methods well known to those skilled in the art. The sample containing the particular proteins to be detected may be obtained from any biological source. Examples of biological sources include blood serum, blood plasma, urine, spinal fluid, saliva, fermentation fluid, lymph fluid, tissue culture fluid and ascites fluid of a human or animal. In a particular example, the biological source is saliva. The sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to assay to optimize the immunoassay results. The fluid migrates distally through all the functional regions of the strip. The final distribution of the fluid in the individual functional regions depends on the adsorptive capacity and the dimensions of the materials used. In some embodiments, porous solid supports, such as nitrocellulose, described hereinabove are preferably in the form of sheets or strips. The thickness of such sheets or strips may vary within wide limits, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such sheets or strips may similarly vary within wide limits, for example from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; however, pore size is not intended to be a limiting factor in selection of the solid support. The flow rate of a solid support, where applicable, can also vary within wide limits, for example from about 12.5 to 90 sec/cm (i.e., 50 to 300 sec/4 cm), about 22.5 to 62.5 sec/cm (i.e., 90 to 250 sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250 sec/4 cm), about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), or about 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specific embodiments of devices described herein, the flow rate is about 62.5 sec/cm (i.e., 250 sec/4 cm). In other specific embodiments of devices described herein, the flow rate is about 37.5 sec/cm (i.e., 150 sec/4 cm). Another common feature to be considered in the use of assay devices is a means to detect the formation of a complex between an analyte (such as one or more proteins described herein) and a capture reagent (such as one or more antibodies). A detector (also referred to as detector reagent) serves this purpose. A detector may be integrated into an assay device (for example included in a conjugate pad, as described below), or may be applied to the device from an external source.

A detector may be a single reagent or a series of reagents that collectively serve the detection purpose. In some instances, a detector reagent is a labeled binding partner specific for the analyte (such as a gold-conjugated antibody for a particular protein of interest, for example those described herein). In other instances, a detector reagent collectively includes an unlabeled first binding partner specific for the analyte and a labeled second binding partner specific for the first binding partner and so forth. Thus, the detector can be a labeled antibody specific for a protein described herein. The detector can also be an unlabeled first antibody specific for the protein of interest and a labeled second antibody that specifically binds the unlabeled first antibody. In each instance, a detector reagent specifically detects bound analyte of an analyte-capture reagent complex and, therefore, a detector reagent preferably does not substantially bind to or react with the capture reagent or other components localized in the analyte capture area. Such non-specific binding or reaction of a detector may provide a false positive result. Optionally, a detector reagent can specifically recognize a positive control molecule (such as a non-specific human IgG for a labeled Protein A detector, or a labeled Protein G detector, or a labeled anti-human Ab(Fc)) that is present in a secondary capture area. Flow-Through Device Construction and Design

A flow-through device involves a capture reagent (such as one or more antibodies) immobilized on a solid support, typically, microtiter plate or a membrane (such as, nitrocellulose, nylon, or PVDF). Characteristics of useful membrane have been previously described; however, it is useful to note that in a flow-through assay capillary rise is not a particularly important feature of a membrane as the sample moves vertically through the membrane rather than across it as in a lateral flow assay. In a simple representative format, the membrane of a flow-through device is placed in functional or physical contact with an absorbent layer (see, e.g., description of "absorbent pad" below), which acts as a reservoir to draw a fluid sample through the membrane. Optionally, following immobilization of a capture reagent, any remaining protein-binding sites on the membrane can be blocked (either before or concurrent with sample administration) to minimize nonspecific interactions.

In operation of a flow-through device, a fluid sample (such as a bodily fluid sample) is placed in contact with the membrane. Typically, a flow-through device also includes a sample application area (or reservoir) to receive and temporarily retain a fluid sample of a desired volume. The sample passes through the membrane matrix. In this process, an analyte in the sample (such as one or more protein, for example, one or more proteins described herein) can specifically bind to the immobilized capture reagent (such as one or more antibodies). Where detection of an analyte-capture reagent complex is desired, a detector reagent (such as labeled antibodies that specifically bind one or more proteins) can be added with the sample or a solution containing a detector reagent can be added subsequent to application of the sample. If an analyte is specifically bound by capture reagent, a visual representative attributable to the particular detector reagent can be observed on the surface of the membrane. Optional wash steps can be added at any time in the process, for instance, following application of the sample, and/or following application of a detector reagent.

Lateral Flow Device Construction and Design

Lateral flow devices are commonly known in the art. Briefly, a lateral flow device is an analytical device having as its essence a test strip, through which flows a test sample fluid that is suspected of containing an analyte of interest. The test fluid and any suspended analyte can flow along the strip to a detection zone in which the analyte (if present) interacts with a capture agent and a detection agent to indicate a presence, absence and/or quantity of the analyte.

Numerous lateral flow analytical devices have been disclosed, and include those shown in U.S. Patent Nos. 4,313,734; 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240; 4,857,453; 4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078; 5,126,241; 5,451,504; 5,424,193; 5,712,172; 6,555,390; 6,258,548; 6,699,722; 6,368,876 and 7,517,699; EP 0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439, each of which is incorporated by reference. Many lateral flow devices are one-step lateral flow assays in which a biological fluid is placed in a sample area on a bibulous strip (though non-bibulous materials can be used, and rendered bibulous, e.g., by applying a surfactant to the material), and allowed to migrate along the strip until the liquid comes into contact with a specific binding partner (such as an antibody) that interacts with an analyte (such as one or more proteins) in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent or otherwise visible dye) indicates that the interaction has occurred. Multiple discrete binding partners (such as antibodies) can be placed on the strip (for example in parallel lines) to detect multiple analytes (such as two or more proteins) in the liquid. The test strips can also incorporate control indicators, which provide a signal that the test has adequately been performed, even if a positive signal indicating the presence (or absence) of an analyte is not seen on the strip.

The construction and design of lateral flow devices is very well known in the art, as described, for example, in Millipore Corporation, A Short Guide Developing Immunochromato graphic Test Strips, 2nd Edition, pp. 1-40, 1999, available by request at (800) 645-5476; and Schleicher & Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp. 73-98, 2003, 2003, available by request at Schleicher & Schuell BioScience, Inc., 10 Optical Avenue, Keene, NH 03431, (603) 352-3810; both of which are incorporated herein by reference. Lateral flow devices have a wide variety of physical formats that are equally well known in the art. Any physical format that supports and/or houses the basic components of a lateral flow device in the proper function relationship is contemplated by this disclosure.

The basic components of a particular embodiment of a lateral flow device are illustrated in FIG. 3A, which shows a particular embodiment of a bibulous lateral flow strip 12. Lateral flow strip 12 is divided into a proximal sample application pad 14, an intermediate test result zone 16, and a distal absorbent pad 18. Flow strip 12 is interrupted by a conjugate pad 19 that contains labeled conjugate (such as gold- or latex-conjugated antibody specific for the target analyte or an analyte analog). A flow path along strip 12 passes from proximal pad 14, through conjugate pad 19, into test result zone 16, for eventual collection in absorbent pad 18. Selective binding agents are positioned on a proximal test line 20 in test result membrane 16. A control line 22 is provided in test result zone 16, slightly distal to test line 20. For example, in a competitive assay, the binding agent in test line 20 specifically binds the target analyte, while control line 22 less specifically binds the target analyte.

In operation of the particular embodiment of a lateral flow device illustrated in FIG. 3A, a fluid sample containing an analyte of interest, such as one or more proteins described herein (for example, AlAT or AlAG, or other combinations of proteins, as discussed above), is applied to the sample pad 14. In some examples, the sample may be applied to the sample pad 14 by dipping the end of the device containing the sample pad 14 into the sample (such as saliva or urine) or by applying the sample directly onto the sample pad 14 (for example by placing the sample pad 14 in the mouth of the subject). In other examples where a sample is whole blood, an optional developer fluid is added to the blood sample to cause hemolysis of the red blood cells and, in some cases, to make an appropriate dilution of the whole blood sample.

From the sample pad 14, the sample passes, for instance by capillary action, to the conjugate pad 19. In the conjugate pad 19, the analyte of interest, such as a protein of interest, may bind (or be bound by) a mobilized or mobilizable detector reagent, such as an antibody (such as antibody that recognizes one or more of the proteins described herein). For example, a protein analyte may bind to a labeled (e.g., gold-conjugated or colored latex particle-conjugated) antibody contained in the conjugate pad. The analyte complexed with the detector reagent may subsequently flow to the test result zone 16 where the complex may further interact with an analyte-specific binding partner (such as an antibody that binds a particular protein, an anti-hapten antibody, or strep tavidin), which is immobilized at the proximal test line 20. In some examples, a protein complexed with a detector reagent (such as gold-conjugated antibody) may further bind to unlabeled, oxidized antibodies immobilized at the proximal test line 20. The formation of a complex, which results from the accumulation of the label (e.g., gold or colored latex) in the localized region of the proximal test line 20 is detected. The control line 22 may contain an immobilized, detector-reagent- specific binding partner, which can bind the detector reagent in the presence or absence of the analyte. Such binding at the control line 22 indicates proper performance of the test, even in the absence of the analyte of interest. The test results may be visualized directly, or may measured using a reader (such as a scanner). The reader device may detect color or fluorescence from the readout area (for example, the test line and/or control line).

In another embodiment of a lateral flow device, there may be a second (or third, fourth, or more) test line located parallel or perpendicular (or in any other spatial relationship) to test line 20 in test result zone 16 (for example test lines 20a, 20b, and 20c in FIG. 3B). The operation of this particular embodiment is similar to that described in the immediately preceding paragraph with the additional considerations that (i) a second detector reagent specific for a second analyte, such as another antibody, may also be contained in the conjugate pad, and (ii) the second test line will contain a second specific binding partner having affinity for a second analyte, such as a second protein in the sample. Similarly, if a third (or more) test line is included, the test line will contain a third (or more) specific binding partner having affinity for a third (or more) analyte.

1. Sample Pad

The sample pad (such as sample pad 14 in FIG. 3A) is a component of a lateral flow device that initially receives the sample, and may serve to remove particulates from the sample. Among the various materials that may be used to construct a sample pad (such as glass fiber, woven fibers, screen, non-woven fibers, cellosic fibers or paper), a cellulose sample pad may be beneficial if a large bed volume {e.g., 250 μl/cm ) is a factor in a particular application. Sample pads may be treated with one or more release agents, such as buffers, salts, proteins, detergents, and surfactants. Such release agents may be useful, for example, to promote resolubilization of conjugate-pad constituents, and to block non-specific binding sites in other components of a lateral flow device, such as a nitrocellulose membrane. Representative release agents include, for example, trehalose or glucose (1% - 5%), PVP or PVA (0.5% - 2%), Tween 20 or Triton X-100 (0.1% - 1%), casein (1% - 2%), SDS (0.02% - 5%), and PEG (0.02% - 5%).

2. Membrane and Application Solution:

The types of membranes useful in a lateral flow device (such as nitrocellulose (including pure nitrocellulose and modified nitrocellulose), nitrocellulose direct cast on polyester support, polyvinylidene fluoride, or nylon), and considerations for applying a capture reagent to such membranes have been discussed previously.

3. Conjugate Pad

The conjugate pad (such as conjugate pad 19 in FIG. 3A) serves to, among other things, hold a detector reagent. Suitable materials for the conjugate pad include glass fiber, polyester, paper, or surface modified polypropylene. In some embodiments, a detector reagent may be applied externally, for example, from a developer bottle, in which case a lateral flow device need not contain a conjugate pad (see, for example, U.S. Patent No. 4,740,468).

Detector reagent(s) contained in a conjugate pad is typically released into solution upon application of the test sample. A conjugate pad may be treated with various substances to influence release of the detector reagent into solution. For example, the conjugate pad may be treated with PVA or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other release agents include, without limitation, hydroxypropylmethyl cellulose, SDS, Brij and β-lactose. A mixture of two or more release agents may be used in any given application. In a particular disclosed embodiment, the detector reagent in conjugate pad 19 is a gold-conjugated antibody. 4. Absorbent Pad

The use of an absorbent pad 18 in a lateral flow device is optional. The absorbent pad acts to increase the total volume of sample that enters the device. This increased volume can be useful, for example, to wash away unbound analyte from the membrane. Any of a variety of materials is useful to prepare an absorbent pad, for example, cellulosic filters or paper. In some device embodiments, an absorbent pad can be paper {i.e., cellulosic fibers). One of skill in the art may select a paper absorbent pad on the basis of, for example, its thickness, compressibility, manufacturability, and uniformity of bed volume. The volume uptake of an absorbent made may be adjusted by changing the dimensions (usually the length) of an absorbent pad.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Materials and Methods

Subjects: From a prospective observational study, 40 subjects were chosen for salivary proteome analysis. In the study group, 10 each had impaired glucose tolerance (IGT), both IGT and impaired fasting glucose (IFG), or (previously diagnosed) diabetes (DM). The diagnosis of DM and pre-diabetes (IFG and IGT) was based on American Diabetes Association criteria. IFG was diagnosed if the fasting plasma glucose level was elevated (between 100 and 125 mg/dl after an overnight fast), and IGT if the 2-hour plasma glucose level was elevated (between 5 140 and 199 mg/dl) after an oral glucose tolerance test. The control group consisted of 10 clinically healthy humans between 36 and 62 years of age. Exclusion criteria for the control group included pregnancy, alcohol consumption, tobacco products (former or current), chronic medical illness, history of drug treatment or therapy within the previous months, or history of diabetes. Subjects were asked not to eat,0 smoke, or drink (except water) for an overnight fast prior to collection of saliva samples. Their diets were similar with respect to protein content and uptake of fat and carbohydrates. Socioeconomic status was similar for all groups (based on survey data). The clinical characteristics of healthy controls and study subjects are shown in Table 4. Informed consent was obtained from the subjects following the5 institutional review board guidelines for human subjects (Nizam's Institute of Medical Sciences, Hyderabad, India).

Table 4. Clinical characteristics of controls and subjects with pre-clinical and type-2 diabetes 0 Data are mean ± SD. NA: not applicable; IFG: impaired fasting glucose; IGT: impaired glucose tolerance; DM: type-2 diabetes.

Controls IGT IFG+IGT DM

Age (years) 4620±11 32 438±13 18 4880±823 4975±789

Duration of DM (years) NA NA NA 400±2 16

Height (cm) 16300±1342 16780±753 16640±404 16850±695

Weight (kg) 7366±17 74 7236±844 71 70±651 71 18±1089

BMI (kg/m²) 2757±447 2581 ±364 2590±1 96 2501 ±276

Waist (cm) 9340±1320 9220±683 9480±661 8875±450

Hip (cm) 9940±684 9520±653 9680±602 9325±602

Waist-to-hip ratio 094±008 097±009 098±002 095±002

Blood pressure, systolic (mmHg) 13540±2980 13560±2725 13940±1746 12700±1695 diastolic (mmHg) 8400±967 8900±11 18 8940±961 7925±1237

Plasma glucose, fasting (mg/dl), 8600±857 9360±669 10640±744 15800±6966

2 hours after oral glucose (mg/dl) 11340±21 03 15480±1923 15980±1672 24800±134 71

Serum cholesterol (mg/dl) 201 80±1397 21520±4276 15360±1599 20275±4532

Triglycerides (mg/dl) 11660±3669 14780±5427 17840±94 11 23225±20561

HDL cholesterol (mg/dl) 4460±21 27 3940±627 3240±4 10 3350±772 Sample collection and processing: Unstimulated saliva (20 ml) from diabetic, pre-diabetic, and control groups was collected. Briefly, at 8 AM (before breakfast), the subjects were asked to rinse their mouths thoroughly with water, then to tilt their heads forward and allow saliva to flow into a sterile container for 5 minutes. These specimens were immediately frozen and stored at -80⁰C until analysis. Saliva samples (10 ml) were centrifuged at 10,000xg for 20 min at 4°C to discard cellular debris and nuclei. The supernatants were transferred to 4-ml Ultrafree 5K membrane concentrators (Millipore, Billerica, MA, USA) and spun at 7000 x g to reduce the volumes to ~1 ml. A total of 5 saliva samples from the control and DM groups were pooled together and subjected to two-dimensional liquid chromatography (2-DLC) and LC-tandem mass spectrometry (LC-MS/MS) analysis as described before (Nagalla et al., J Proteome Res 6:1245-1257, 2007) and briefly outlined below.

2-DLC sample processing: Following protein assay, 1-mg portions of samples were reduced, alkylated, digested with trypsin, and the resulting peptides separated with strong cation-exchange (SCX) chromatography. SCX chromatography was performed using a 100 x 2.1 -mm polysulfoethyl A column (The Nest Group, Southborough, MA). A total of 80 fractions were collected and desalted using a 96- well Vydac Cl 8 silica spin plate (The Nest Group). The desalted fractions were consolidated into 31 fractions, dried, dissolved in 20 μl of 5% formic acid for LC-MS/MS analysis.

LC-MS/MS analysis: Portions of each fraction (9 μl) were analyzed by LC- MS/MS using an Agilent 1100 series capillary LC system and an LTQ ion-trap mass spectrometer (Thermo Electron, San Jose, CA) with an Ion Max electrospray source (ThermoFinnigan, San Jose, CA). Samples were applied at 20 μl/min to a trap cartridge, and then switched onto a 0.5 x 250-mm Zorbax SB-C18 column (Agilent Technologies, Palo Alto, CA) using mobile phase A containing 0.1% formic acid. Survey MS scans were alternated with three data-dependent MS/MS scans using the dynamic exclusion feature of the control software to increase the number of unique peptides analyzed. Mass spectra files were generated using Bioworks Browser software (version 3.1, ThermoFinnigan, San Jose, CA) with m/z range of 400-4000 Da, a minimum of 15 ions, and a low TIC threshold of 500. A total of 1,729,998 tandem mass spectra were generated from all LC-MS/MS analyses.

Peptide and protein identification: Tandem mass spectra were searched against a composite protein database containing forward and reversed entries (decoy proteins) of Swiss-Prot (version 52.1) and TrEmbl (version 35.1) databases selected for human subspecies. Splice variants were generated using the varsplice program from the SwissKnife package (version 1.62). Forward and reverse entries of the generated splice variants were also added to the composite protein database. All searches were performed using the X! Tandem (Craig et al., Bioinformatics 20:1466- 1467, 2004) search engine configured to use 1.8 Da and 0.4 Da as parent and fragment ion mass tolerances, respectively, trypsin enzyme specificity, a fixed carbamidomethyl modification on cysteine residues, and several potential in vivo modifications. Peptide identifications from samples were assembled into proteins using probabilistic protein identification algorithms (Nesvizhskii et al., Anal Chem 75:4646-4658, 2003) implemented in Scaffold software (version 1.6, Proteome Software, Portland, OR).

Peptide and protein identifications in all samples were compiled together to generate a comprehensive diabetic salivary proteome. Proteins with one or more unique peptide identifications (p > 0.8) were considered as likely to be present in the sample. Protein entries were further curated to reduce redundancy by removing subset proteins and collapsing degenerate protein identifications into a single entry. All immunoglobulin variants identified in the sample were also collapsed into a single entry. Annotations of identified hypothetical sequences were corrected, if possible, by checking their sequence homology with known proteins in the Swiss- Prot human database (version 52.1) using NCBI BLAST software. A hypothetical annotation was accepted into the final protein list if the corresponding blast search did not turn up any homologous (> 90% sequence homology) known human proteins. Protein identifications with at least three unique peptide identifications in at least one sample were considered to be present in saliva. Label-free quantification: The total number of tandem mass spectra matched to a protein (spectral counting) is a label-free, sensitive, and semi-quantitative measure for estimating its abundance in complex mixtures (Liu et al., Anal. Chem. 76: 4193-4201, 2004; Old et al., MoI. Cell Proteomics 4: 1487-1502, 2005). The spectral count difference between two complex samples is used to quantify the relative expression of a protein (Nagalla et al., J. Proteome Res. 6: 1245-1257, 2007). In this study, salivary proteins with at least three unique peptide identifications in at least one sample were considered for label-free quantification. Shared spectral counts of non-degenerate proteins belonging to the same family with significant sequence homology (>50%) were combined into single entry. Shared spectral counts of non-degenerate proteins that did not fit the afore-mentioned criteria were assigned to one of the proteins using Occam's razor approach. Curated proteins were subjected to independent pair- wise comparisons to determine differentially abundant proteins between control and diabetes groups using either a 2x2 χ² or Fisher's exact test. Normalization of spectral counts to account for experimental variability was built into the pair-wise comparison model automated using a SAS program (version 9.1). A protein was considered as significantly differentially abundant between the samples if the comparison had a p- value of < 0.05 in either the χ or Fisher's exact test. The fold change (FC) in the level of differentially abundant proteins was calculated using the equation described by Old et al. (Old et al., MoI. Cell Proteomics 4: 1487-1502, 2005). Western immunoblotting: Fifteen micrograms of saliva protein from each experimental group (CTRL, IGT, IGT+IFG, and DM) were resolved on 10-20% Tris-Tricine gels and transferred to PVDF membranes. Membranes were blocked with 5% fat-free milk in TBST for 2 hours at room temperature and incubated with primary antibody (alpha- 1 -antitrypsin, cystatin C, alpha-2-macroglobulin, and transthyretin from Dako North America Inc., Carpinteria, CA; and salivary alpha amylase from Sigma, Atlanta, GA) for 1 hour. All primary antibodies were diluted 1:4000, except alpha- 1 -antitrypsin (AlAT), which was diluted 1:5000. After three 10-minute washes with TBST, membranes were incubated with anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalog # SC2004) for 1 hour, washed again in TBST, and then visualized with the

SuperSignal West Pico chemiluminescent substrate system (Pierce, Rockford, IL). Chemiluminescence was scanned on a LAS-3000 instrument using the LAS-3000 Lite software, and scanned images were visualized and quantified using MultiGauge v.3.1 software (FujiFilm Life Science, Inc, Stamford, CT).

ELISAs were performed individually on 5 subjects in each group using a microtiter plate assay. Primary, secondary antibodies, and reference proteins were obtained from Dako North America, Inc. A standard curve was generated by four- parameter curve-fitting using SoftmaxPro V 1.11 software, (Molecular Devices Corporation). The concentrations of the individual samples were estimated from the average values of triplicates in comparison to the standard curve. Concentrations of individual biomarkers are expressed as means +/- SEM. Statistical significance was estimated by Kruskal-Wallis nonparametric ANOVA for 4-group comparison and the Wilcoxon two-sample test for pair-wise comparisons.

Example 2 Human diabetic salivary proteome

A total of 2172 proteins were identified at a single unique-peptide (p > 0.8) threshold. To reduce the false-positive rate, a stringent three-unique-peptide threshold was adapted which resulted in 586 identifications with a false-positive rate of 0.5%. The protein list was further curated by collapsing all the immunoglobulin entries into a single entry, and sample processing artifacts (such as trypsin and keratin) and decoy proteins were removed. The resulting 487 proteins of the salivary proteome and their corresponding spectral count in control and diabetes subject was evaluated. Salivary proteomes from this study and the current literature (Hu et al., Proteomics 5:1714-1728, 2005; Hu et al., Expert Rev Proteomics 4:531- 538, 2007; Vitorino et al., Proteomics 4:1109-1115, 2004; Wilmarth et al., J

Proteome Res 3:1017-1023, 2004) were cross-referenced and marked accordingly in Supplemental Table 1 (Rao et al., J. Proteome Res. 8:239-245, 2009, incorporated herein by reference). A total of 315 (67%) proteins found in this study were confirmed by other studies. Thus, 33% of the salivary proteins identified were newly identified. The type-2 diabetes salivary proteome was functionally annotated using GO annotations from DAVID and BioHarvester informatics resources as shown in FIG. 1. A majority of the salivary proteins have metabolic (42%) and immune response (11%) functions. Proteins with other cellular functions included such as cell organization and biogenesis (11%), cell communication and proliferation (6%), development (5%), and apoptosis (5%).

Example 3 Quantification of diabetic salivary proteome using spectral counts.

The spectral counts of the salivary proteins were subjected to label-free quantification to find differentially abundant proteins between the control and diabetes groups. Proteins with a relative differential abundance of >2.0 fold and which passed the label-free quantification with a p- value of <0.05 were considered as significantly differentially abundant between the two groups. A total of 65 differentially abundant salivary proteins are shown in Table 5 grouped according to their functional annotations. Spectral counts of human salivary proteins with three or more unique peptide identifications were subjected to label-free quantification and those that were significantly differentially abundant (p- value < 0.05) by at least ± 2.0-fold are shown in Table 5.

Table 5. Proteins differentially present in saliva in subjects with type-2 diabetes and controls

Function Swiss- Description Fold P-value

Prot Change

Accession

Diabetes vs. Control

Metabolism P23280 Carbonic anhydrase 6 3.84 <0.0001

P14618 Pyruvate kinase isozymes M1 /M2 3.47 0.0002

P06737 Glycogen phosphorylase, iver 3.32 0.0105 form

Q549C7 Transthyretin 2.4 0.0246

P22894 Neutrophil collagenase 2.36 0.0039

P00491 Purine nucleoside phosphorylase -2.08 0.0032

060235 Transmembrane protease, serine -2.13 0.012 1 1 n

P30838 Aldehyde dehydrogenase, dimeric -2.19 0.0034

NADP-preferring Function Swiss- Description Fold P-value

Prot Change

Accession

Diabetes vs. Control

Q13231 -3 lsoform 2, 3 and 4 of -2.2 0.0263

Chitotriosidase-1

Q9UBR2 Cathepsin Z -2.85 0.0361

P00558 Phosphoglycerate kinase 1 -3.18 <0.0001

060218 Aldo-keto reductase family 1 -3.32 0.0127 member B10

Q13787 Apolipoprotein B-100 -4.13 <0.0001

P00915 Carbonic anhydrase 1 -4.36 <0.0001

P00918 Carbonic anhydrase 2 -5.54 0.0002

Q86U62 Proteasome (prosome, macropain) -6.1 1 0.0184 subunit, beta type, 7

P27824 Calnexin -7.74 0.0005

Immune response Q6FHH3 Uteroglobin 10.43 <0.0001

Q4VAX6 Serpin peptidase inhibitor, clade B 6.05 0.0101

Q9NP55 Protein Plunc 5.48 <0.0001

P13671 Complement component C6 4.75 0.036

P01009 Alpha-1 -antitrypsin 3.24 <0.0001

P01034 Cystatin-C 2.22 0.0007

P30740 Leukocyte elastase inhibitor 2.03 0.01 1

P01040 Cystatin-A -2.42 0.0042

P04083 Annexin A1 -3.57 <0.0001

Development Q4VB24 Histone cluster 1 , H1 e 6.05 0.0101

Q09666 Neuroblast differentiation- 3.08 0.0472 associated protein AHNAK

Q9NZT1 Calmodulin-like protein 5 -2.17 0.0151

Q01469 Fatty acid-binding protein, -2.55 <0.0001 epidermal

Q06830 Peroxiredoxin-1 , -2 and -6 -2.59 <0.0001

Q96RM1 Small proline-rich protein 2F -2.85 0.0361

P31 151 Protein S100-A7 -2.94 0.003

Q5TCI8 Lamin A/C -3.26 <0.0001

P07355 Annexin A2 -4.25 0.0014

P 15924 Desmoplakin -5.88 <0.0001

P30043 Flavin reductase -6.1 1 0.0003

Extracellular matrix P07998 Ribonuclease pancreatic 3.78 0.0015

Protein A2RTY6 Inter-alpha (Globulin) inhibitor H2 3.16 0.0102

P19827 Inter-alpha-trypsin inhibitor heavy 2.8 0.0042 chain H1

P36222 Chitinase-3-like protein 1 2.65 0.0173

Q14624 Inter-alpha-trypsin inhibitor heavy 2.59 0.006 chain H4

P80303 Nucleobindin-2 2.05 0.005

Q9UKR3 Kallikrein-13 -4.48 0.0265 Function Swiss- Description Fold P-value

Prot Change

Accession

Diabetes vs. Control

043240 Kallιkreιn-10 -4.99 0.0024

Signal Q7M4Q5 Basic prohne-πch peptide IB-8a 5.4 0.019 transduction P39687 Acidic leucine-πch nuclear 3.32 0.0105 phosphoprotein 32 family

Q5VY30 Retinol binding protein 4, plasma 2.15 0.0143

P23528 Cofιlιn-1 2.1 1 0.0464

P62258 14-3-3 protein epsilon -2.25 0.01

P 12429 Annexin A3 -2.68 0.008

Q04917 14-3-3 protein eta -2.95 0.0438

Cell organization O1551 1 Actin-related protein 2/3 complex 6.05 0.0101 and biogenesis subunit 5

P60953-2 lsoform 2 of P60953 Cell division 4.75 0.036 control protein 42 homolog precursor

P01023 Alpha-2-macroglobulιn 2.23 <0.0001

P28676 Grancalcin -7.09 0.0083

Cell motility P61 160 Actin-like protein 2 3.36 0.0476

P26038 Moesin 2.04 0.0006

O95274 Ly6/PLAUR domain-containing -2.3 0.0236 protein 3

P67936-2 lsoform 2 of P67936 Tropomyosin -3.75 0.0006 alpha-4 chain

Spectral counts of human salivary proteins with 3 or more unique peptide identifications were subjected to label-free quantification Proteins that were significantly differentially abundant (p- value < 0 05) by at least ± 2 0-fold are shown above Proteins are grouped according to their function Fold change between the groups was quantified using equation described by Old et al (MoI Cell Proteomics 2005, 4, (10), 1487-502)

Example 4 Validation of potential biomarkers using immunodetection.

To independently confirm the relative abundance of salivary proteins identified by 2D-LC-MS analysis, immuno-quantification was performed using western blotting. To further explore their potential performance in pre-diabetes, samples were tested from IGT and IGT+IFG groups in addition to type-2 diabetes. As shown in Figure 2, the levels of AlAT, cystatin C (CysC), alpha-2- macroglobulin (A2MG), and transthyretin (TTR) were elevated in type-2 diabetes, which correlated with the results of spectral counting. Scanning densitometry analysis showed the relative levels of A2MG as 1.4, 1.9, and 2.1, AlAT as 1.4, 2.0, and 2.5, and TTH as 1.26, 1.38 and 1.68-fold higher in the IGT, IGT+IFG, and type- 2 diabetes groups, respectively, compared to control (1.0). As also shown in Figure 2, AlAT, A2MG, and TTR showed a relative increase in expression with disease progression. In contrast, the relative levels of CysC were 1.54, 1.28, and 1.29-fold higher in the IGT, IGT+IFG, and type-2 diabetes groups, respectively, compared to control; i.e., highest in the IGT group.

Immunoassay measurements of A2MG on individual subjects correlated with the western blot data and their differential abundance based upon label-free quantification (FIG. 2). There were significant differences in A2MG concentrations in the 4 groups when compared via Kruskal-Wallis nonparametric ANOVA (p=0.0186 for the 4-group comparison). In pair-wise comparisons, the group significantly different from control was the DM group (p=0.0137 via Wilcoxon two- sample test). These data suggest that the differential abundance of specific salivary proteins in DM saliva is potentially presaged by their differential abundance in pre- diabetes.

Diabetes is a major problem worldwide and is a leading cause of morbidity that is attributable to largely preventable metabolic complications. To date, however, no robust marker of diabetes or its vascular complications has been validated for general clinical use. With the emergence of disease-modification drugs in diabetes, there is an increasing need for diagnostic markers to ensure that these therapies are targeted to the correct patient population.

Saliva has multiple advantages as a diagnostic body fluid due to its noninvasive, safe, simple, and cost-effective nature. By using a comprehensive and rigorous proteomic approach comprised of 2-DLC fractionation, LC-MS/MS identification, and spectral counting quantification, 487 proteins were characterized in human whole saliva and 65 were identified that were significantly different in relative abundance between controls and type-2 diabetes patients. The majority of the differently abundant proteins are predicted to have functions in metabolism, followed by the functional categories of development, cell organization and biogenesis, immune function, cell communication and proliferation, and apoptosis. Among the up-regulated proteins identified in this study are proteins associated with immune function that have been previously reported to be associated with diabetes in other body fluids. The presence of inflammatory factors among this biomarker set is consistent with the proposed role of a chronic sub-clinical inflammatory state in the genesis of the metabolic syndrome and diabetes. The protease inhibitors CysC, leukocyte elastase inhibitor (LEI), and uteroglobin also have individually be associated with diabetes. CysC is a potent inhibitor of lysosomal cysteine proteinases. In serum, it is a known marker for glomerular filtration and has been shown to be significantly elevated in cardiovascular disease and diabetes (Larrson et al., Int J Cardiol, 2007). LEI regulates the activity of neutrophil proteases, including polymorphonuclear elastase (PMN-E). In human plasma, PMN-E is a marker for hypertension and micro- and macro-vascular disease in type-2 diabetes (Piwowar et al., Clin Chem Lab Med 38:1257-1261, 2000). Human uteroglobin, also called blastokinin, is secreted from Clara cells (nonciliated cells of the surface epithelium of the pulmonary airways). In human urine, it is a known indicator of renal tubular function in diabetes (Hong et al., J Diabetes Complications 12:43-60, 1998). Neutrophil collagenase or MMP-8 has been linked to local tissue damage rather than to neutrophil dysfunction in saliva from patients with uncontrolled type-2 diabetes (Collin et al., J Periodontal Res 35:259-265, 2000). However, serum MMP-8 levels were reported to be elevated in coronary heart disease independent of known risk factors, including diabetes (Qiang et al., Nan Fang Yi Ke Da Xue Xue Bao 27:831-833, 2007). Thus, up-regulated MMP-8 in human saliva in type-2 diabetes could be a non-specific inflammatory marker. TTR, or prealbumin, is a known transport protein for both thyroxine and retinol (vitamin A). Stockholm Diabetes Prevention Program investigators have reported both up- and down-regulation of serum TTR, depending on the particular patient cohort studied (Sundsten et al., Diabetes Metab Res Rev, 2007)

It is disclosed herein that AlAT, A2MG and plasma retinol binding protein 4 (RBP4) were elevated in diabetic saliva. These proteins have been implicated in diabetes. AlAT, potentially through its demonstrated anti-apoptotic activity, has also been shown to prevent or reverse diabetes, prevent the development of type-1 diabetes in mice, prolong islet allograft survival in rodents, and reduce beta-cell apoptosis in vitro (Zhang et al., Diabetes 56: 1316-1323, 2007). Over-expression of AlAT has been seen in the urine of patients with diabetic nephropathy. RBP4 is a novel adipokine of the lipocalin family involved in the development of obesity and insulin resistance (Rao et al., Diabetes Care 30:629-637, 2007). Its abundance in human saliva in the present study was consistent with elevated RBP4. A2M variations in diabetes were reported with protease-antiprotease imbalance in children who were at greater risk of developing vascular complications ( Lisowska-Myjak et al., Acta Diabetol 43:88-92, 2006). A cardiac isoform of A2MG has been shown to be an early marker of cardiac hypertrophy and increased left-ventricular mass in myocardial-infracted diabetic patients (Annapoorani et al., Atherosclerosis 186:173- 176, 2006), and A2MG in human saliva was reported as a proinflammatory factor (Aurer et al., Coll Antropol 29:435-439, 2005). Carbonic anhydrase I (CA-I) was down-regulated in saliva, and was reported to be decreased in erythrocytes (Gambhir et al., Biochem Genet 45:431-439, 2007) and increased in vitreous of type-2 diabetes patients. Apolipoprotein (apo) B-100, is one of the two main forms of apoB in chylomicrons and low-density lipoproteins. Diabetic subjects are known to have elevated apoB-100 as a result of increased production and reduced fractional catabolic rates (Hogue et al., J Lipid Res 48: 1336-1342, 2007). One of the nine development-related salivary proteins down-regulated in type-2 diabetes was lamin A/C. Mutations in the LMNA gene are characterized by loss of subcutaneous adipose tissue, insulin resistance, dyslipidemia, and type-2 diabetes (Hegele et al., J Clin Endocrinol Metab 92:4566-4568, 2007).

The studies disclosed herein established a subset of salivary biomarkers of established type-2 diabetes identified by proteomic profiling. These biomarkers were differentially abundant in the saliva of patients with IGT alone and IGT + IFG as assessed by direct Western immunoblot analysis. The relative increase of some of these markers in association with progression of pre-diabetes to the diabetic state underscores the importance of a systematic analysis of these candidate biomarkers in pre-diabetic saliva, as well as their variability in individual samples, by immunoassays. As recent studies have shown that early and multi-factorial intervention in type-2 diabetes prevents cardiovascular complications and mortality, accurate diagnosis of this condition facilitates early intervention.

Example 5 Additional biomarkers for Pre-diabetes and Diabetes

Additional salivary glycoprotein markers of pre-diabetes and diabetes were evaluated using an ELISA assay. A cohort of 159 subjects including controls (OGTT<140 mg/dl), pre-diabetes (OGTT 140-199 mg/dl), and type 2 diabetes (OGTT >199 mg/dl) were tested for the presence of alpha 1 acid glycoprotein (AlAG) and lipocalin 2 in saliva samples.

There were significant differences in both AlAG and lipocalin 2 concentrations in between control and pre-diabetes and diabetes groups when compared via one way analysis of variance using Dunnett's post-hoc correction factor for pairwise comparisons versus control (Table 6).

Table 6. Protein Concentration in Saliva Samples from Subjects with Prediabetes or Diabetes

ne-way analysis of variance using Dunnett' s post-hoc correction factor for pairwise comparisons versus the control

Example 6 Exemplary Diagnostic Study

A subject presents with a BMI greater than or equal to about 30 kg/m² and a family history of diabetes. The subject reports a sedentary lifestyle. A saliva sample is obtained from the subject. An ELISA is performed on the saliva sample, and the amount of at least one protein listed in Table 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 proteins is determined relative to amount of these proteins in a saliva sample from a subject without diabetes. The subject is identified as having at least one such protein altered as compared to a control. The subject is identified with relative levels of one such protein as at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5-fold higher than the control. For example, the subject is identified with relative levels of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter- α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin as 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5-fold altered as compared to the level of the protein in the control. Thus, the subject is identified as pre-diabetic. The amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is also determined relative to the amount of these proteins in a saliva sample from a subject known to be diabetic. A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is altered when compared to the amount of these proteins in the saliva sample from the diabetic subject. This confirms that the subject is pre-diabetic, and does not have frank type 2 diabetes. Thus, the method can be used to distinguish pre-diabetes from diabetes.

Example 7 Exemplary Diagnostic Study A subject presents with FPG of 100 mg/dl and a OGTT of 140 mg/dl. A saliva sample is obtained from the subject. An ELISA is performed on the saliva sample, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is determined relative to amount of these proteins in a saliva sample from a control subject without diabetes. The amount of at least one of the proteins set forth in Table 2 is also determined relative to the amount of this protein in a saliva sample from the control. Hemoglobin AlC is also assessed. The subject is identified as having all of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin altered as compared to a control. The subject is identified with relative levels of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin as 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5-fold higher than the control. The amount of at least one of the protein set forth in Table 2 also differs 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or 3.5-fold from the control.

A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin compared to the amount of these proteins in the saliva sample from the normal subject. Both the glycosylated form and the unglycosylated form of the protein are measured. Thus, the subject is identified as pre-diabetic. The amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is also determined relative to the amount of these proteins in a saliva sample from a subject known to be diabetic. A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is altered from the amount of these proteins in the saliva sample from the diabetic subject. This confirms that the subject is pre-diabetic.

Example 8 Evaluating Progression of Diabetes A subject presents with FPG of 120 mg/dl and a OGTT of 190 mg/dl. The subject is treated with metformin. After one month of treatment, a saliva sample is obtained from the subject. An ELISA is performed on the saliva sample, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is determined relative to amount of these proteins in a saliva sample from a control subject without diabetes.

A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is determined and is compared to the amount of these proteins in the saliva sample from the control subject. Following treatment, the subject is identified as having an amount of at least one of protein plunc, pancreatic ribonuclease, inter-α- trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin that does not significantly differ from the amount of the protein in the saliva sample from the control.

Thus, the therapy is identified as effective for the treatment of the subject. The subject maintains the therapy for an additional year, and a second saliva sample is obtained from the subject. An ELISA is performed on the saliva sample, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is determined and is compared to amount of these proteins in a saliva sample from a control subject without diabetes.

A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is not increased relative to the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin to compare the amount of these proteins in the saliva sample from the control subject. The subject is identified as having an amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin that does not significantly differ from the amount of the protein in the saliva sample from the control. Thus, the therapy is continued for an additional year.

A third saliva sample is obtained from the subject. An ELISA is performed on the saliva sample, and the amount of protein plunc, pancreatic ribonuclease, inter- α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is determined. A statistical analysis is performed, and the amount protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is altered relative to the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin in the saliva sample is compared to the amount in the saliva from a control subject. The amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin is also determined relative to the amount of these proteins in a saliva sample from a subject known to be diabetic. A statistical analysis is performed, and the amount of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α- trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin does not differ significantly from than the amount of these proteins in the saliva sample from the diabetic subject. This confirms that the therapy is no longer effective in treating the subject. The subject is started on insulin therapy and is instructed to make further lifestyle modifications.

Example 9 Exemplary Lateral Flow Device Diagnostic Tests

This example describes exemplary lateral flow devices for diagnosis of prediabetes or diabetes, such as sandwich immunoassay and competitive immunoassay formats. Sandwich Immunoassay Format FIG. 4 schematically illustrates an exemplary lateral flow device for diagnosis of pre-diabetes or diabetes utilizing a sandwich immunoassay format. As in the prior embodiment of FIG. 3A, a labeled first specific binding pair (SBP) member is diffusively bound on the matrix on a conjugate pad 19 at a point upstream of the test result zone 16. The sample is added to a sample application pad 14 on the matrix at a point upstream of the labeling zone and allowed to flow through the labeling zone. The labeled first SBP member located within the conjugate pad is capable of being freely moblizible in the sample. Therefore, if analyte is present in the sample, the labeled first SBP member binds to the analyte and the resulting analyte-labeled first SBP member complex is transported to and through the test result zone. The extent of complex formation between the analyte and the labeled SBP member is directly proportional to the amount of analyte present in the sample. A second SBP member capable of binding to the analyte-first SBP member complex is immobilized on the test result zone. This second SBP member is not capable of binding the labeled first SBP member unless the labeled first SBP member is bound to the analyte. Thus, the amount of labeled SBP member that accumulates on the test result zone is directly proportional to the amount of analyte present in the sample.

In the exemplary device (FIG. 4), the conjugate pad 19 includes a first SBP reagent 40 (such as a first AlAT antibody covalently attached to blue latex particles) and a first test line 20 that includes a second AlAT antibody that recognizes a different epitope of AlAT than the first AlAT antibody. The conjugate pad 19 also includes a second SBP reagent 42 (such as a first AlAG antibody covalently attached to blue latex) and the second test line 22 includes a second AlAG antibody that recognizes a different epitope than the first AlAG antibody.

The test is performed by applying a sample (such as saliva) from a subject to the sample application pad 14. The sample flows through the conjugate pad 19, releasing the first SBP reagent and releasing the second SBP reagent. The displaced first SBP reagent is captured by the first test line 20 if AlAT is bound to the first SBP. The displaced second SBP reagent is captured by the second test line 22 if AlAG is bound to the second SBP. The first and second test line intensity is compared a control (for example, visually or using a reader). If the first or second test line intensity (or both) is greater than the control, then the subject has prediabetes or diabetes. If the first or second test line intensity (or both) is less than the control, then the subject is normal. Competitive Immunoassay Format In another embodiment, the lateral flow device for the diagnosis of prediabetes or diabetes uses a competitive immunoassay format. FIG. 5 shows strip 120 to include an elongated, narrow, bibulous liquid collection member 122 with a flat proximal edge 124 and a flat distal edge 126. Strip 120 is mounted on a rigid or semi-rigid plastic support 128, and a proximal absorbent sample collection pad 130 is also mounted to the support 128 such that it is contiguous with collection member 122 through portion 132. A distal reservoir pad 134 is attached to a distal end of support 128. Liquid (such as a biological fluid, for example, saliva) placed on collection pad 130 moves by capillary action in a distal direction 138 through collection member 122 into reservoir pad 134. Capture agents (such as specific binding partners, for example antibodies such as monoclonal antibodies) are aligned in spaced indicator lines 150, 152, each of which extends transversely on the strip, and respectively form the primary and secondary capture zones. A mobilization zone 154 is located on collection member 122 underneath pad 130 and indicator line 150. The mobilization zone 154 contains an analyte (or analyte analog, for example AlAT and/or AlAG) linked to a label, such as a colored latex microsphere (referred to as an A-L-T conjugate). In this embodiment, the fluid sample (such as saliva) is applied to sample pad 130, which mobilizes the A-L-T conjugate in mobilization zone 154. The A-L-T conjugate moves with the liquid sample through pad 130 and contiguous portion 132.

Since the sample is applied to the surface of pad 130, it is designed to encounter less resistance and migrate more quickly through pad 130 than the subjacent larger or heavier A-L-T conjugate (which must be hydrated and mobilized), so that the sample (and any analyte in the sample) therefore the primary capture line 150 before the A-L-T conjugate. If the analyte is present and the A-L-T conjugate reaches the antibodies (such as anti-AlAT and/or anti-AlAG) in primary capture line 150, the specific binding sites are already occupied by any analyte (for example, AlAT and/or AlAG) from the sample, which reduces the number of binding sites available to bind A-L-T. Hence the A-L-T conjugate continues to migrate by capillary action through the porous material of collection member 122 until it reaches secondary capture line 152, where it is bound by the specific binding partner immobilized therein (for example, strep tavidin). The presence of analyte in the sample is detected by a positive signal (such as a color change from the colored latex microsphere) in secondary capture line 152. If a positive signal is present at secondary capture line 152, then the result indicates that subject has pre-diabetes or diabetes. If analyte is not present in the sample (or is present below a pre-selected threshold) then in the absence of competition to A-L-T binds to primary capture line 150 without migrating in substantial amount to secondary capture line 152. The absence of a significant positive signal (such as a color change) at secondary capture line 152 indicates that the subject does not have pre-diabetes or diabetes.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A method for determining if a subject of interest has pre-diabetes or diabetes, is at risk for developing pre-diabetes or diabetes, or monitoring the efficacy of a therapy, comprising

(a) comparing a proteomic profile of a test sample of saliva from a subject of interest with a proteomic profile of a reference sample, wherein the proteomic profile comprises at least one unique expression signature characteristic of prediabetes or diabetes, respectively, wherein the test sample proteomic profile and the proteomic profile of the reference proteomic sample comprise information on the expression of at least one of alpha- 1 -antitrypsin, alpha 1 acid glycoprotein, cystatin C, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, neutrophil collagenase, alpha 2-macroglobulin, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-100, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, and lipocalin 2, and wherein:

(i) if the reference sample is a normal sample, and the proteomic profile of the test sample is essentially the same as the proteomic profile of the normal sample the subject is determined not to have pre-diabetes or diabetes, respectively, not to be at risk for developing pre-diabetes or diabetes, or to have an effective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the proteomic profile of the normal sample the subject is determined to have prediabetes or diabetes, respectively, to be at risk for developing pre-diabetes or diabetes, or to have an ineffective therapy; (ii) if the reference sample is a sample from a subject with pre-diabetes or diabetes, and proteomic profile shares at least one unique expression signature characteristic with the reference sample then the subject is determined to have prediabetes or diabetes, respectively, to be at risk for developing pre-diabetes or diabetes, or to have an ineffective therapy, while if the proteomic profile of the test sample has a unique expression signature relative to the reference sample the subject is determined not to have pre-diabetes or diabetes, respectively, not to be at risk for developing pre-diabetes or diabetes, or to have an effective therapy.

2. A method for determining if a subject of interest has pre-diabetes or diabetes, is at risk for developing pre-diabetes or diabetes or monitoring the efficacy of a therapy, comprising

(a) comparing a proteomic profile of a test sample of saliva from a subject of interest with a proteomic profile of a reference sample, wherein the proteomic profile comprises at least one unique expression signature characteristic of pre- diabetes or diabetes, respectively, wherein the test sample proteomic profile and the proteomic profile of the reference proteomic sample comprise information on the expression of at least one of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isoform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin, and wherein:

3. The method of claim 2, wherein the proteomic profile further comprises information on the expression of one or more of alpha-1-antitrypsin, alpha 1 acid glycoprotein, cystatin C, uteroglobin, carbonic anhydrase 6, pyruvate kinase isozymes M1/M2, neutrophil collagenase, alpha 2-macroglobulin, purine nucleoside phosphorylase, aldehyde dehydrogenase, fatty acid biding protein (epidermal), peroxiredoxin-1, -2, + -6, lamin A/C, apolipoprotein B-IOO, annexin A2, carbonic anhydrase 1, carbonic anhydrase 2, and lipocalin 2.

4. The method of claim 1 or claim 2, wherein the proteomic profile further comprises information on the expression of one or more of proteasome subunit, aldo-keto reductase family 1 member BlO, cathepsin Z, chitotriosidase isoform 2, 3, + 4, transmembrane protease, serine HD, transthyretin, glycogen phosphorylase, heterogeneous nuclear RNPs A2/B1, leukocyte elastase inhibitor, small proline-rich protein 2F, calmodulin- like protein 5, neuroblast differentiation AHNAK, histone cluster 1, HIe, kallikrein-13, chitinase-3-like protein 1, inter-alpha (Globulin) inhibitor H2, 14-3-3 protein eta, cofilin-1, retinol binding protein 4, plasma, basic proline-rich peptide lB-8a, isoform 2 of P60953 cdc 42 homolog, actin-related protein 2/3 complex subunit 5, Iy6/PLAUR domain-containing protein 3, actin-like protein 2, Rearranged VKA17 V gene segment, brain acid soluble protein 1, golgi phosphoprotein 2, protein FAM49B (Ll), and acidic leucine-rich nuclear phosphoprotein 32.

5. The method of claim 2, wherein the proteomic profile comprises information on the expression of five or more of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter- α- trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

6. The method of claim 2, wherein the proteomic profile comprises information on the expression of ten or more of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter- α- trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

7. The method of claim 2, wherein the proteomic profile comprises information on the expression of fifteen or more of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein SlOO- A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

8. The method of claim 2, wherein the proteomic profile comprises all of protein plunc, pancreatic ribonuclease, inter-α-trypsin inhibitor heavy chain Hl, inter-α-trypsin heavy chain H4, nucleobindin-2, moesin, 14-3-3- epsilon, cystatin A, annexin A3, Protein S100-A7, phosphoglycerate kinase 1, annexin Al, isotform2 of P67936 tropomyosin α-4, kallikrein-10, desmoplakin, flavin reductase, grancalcin, and calnexin.

9. The method of claim 2, wherein the proteomic profile further comprises information on the expression of apha-1 -antitrypsin, alpha 2-macroglobulin, cystatin C, and transthyretin.

10. The method of claim 1 or claim 2, wherein the diabetes is type 2 diabetes.

11. The method of claim 1 or claim 2, wherein the diabetes is type 1 diabetes.

12. The method of claim 1 or claim 2, wherein the method is a method for monitoring the efficacy of therapy.

13. The method of claim 1 or claim 2, further comprising detecting hemoglobin AlC.

14. The method of claim 1 or claim 2, wherein the subject is obese.

15. The method of any of claims 1 to 14, wherein the proteomic profile is determined using a lateral flow device.