US 20120208218A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0208218 A1 Nagalla et al. (43) Pub. Date: Aug. 16, 2012

(54) METHODS FOR DETECTING (30) Foreign Application Priority Data PRE-DABETES AND DABETES USING DIFFERENTIAL PROTEIN GLYCOSYLATION May 11, 2010 (US) ...... PCT/US2O1 O/O34385 Publication Classification (75) Inventors: Srinivasa R. Nagalla, Hillsboro, (51) Int. Cl POAfOR (US); ORCharles ar's T. RobertOperts, GOIN 33/566 (2006.01) s (52) U.S. Cl...... 435/7.94; 436/501; 435/7.92 (73) Assignee: DiabetOmics, LLC, Beaverton, OR (57) ABSTRACT US (US) Methods for identifying individuals who are not yet diabetic (21) Appl. No.: 13/457,243 (pre-diabetic), but who are at significant risk of developing diabetes, such as type 2 diabetes, are disclosed herein. Meth (22) Filed: Apr. 26, 2012 ods are also provided for the identification of diabetic sub 9 jects. Also disclosed are methods for identifying individuals Related U.S. Application Data with diabetic complications. The methods include the identi AV fication of an overall glycosylation profile of proteins in a (62) Division of application No. 12/777,955, filed on May biological fluid, such as saliva, urine, or serum. In some 11, 2010, now Pat. No. 8,168,396. examples, the methods include determining the amount of one or more protein in a biological fluid or determining the (60) Provisional application No. 61/177,130, filed on May glycosylation pattern of one or more proteins in a biological 11, 2009. fluid. Patent Application Publication Aug. 16, 2012 Sheet 1 of 7 US 2012/0208218A1

Patent Application Publication Aug. 16, 2012 Sheet 2 of 7 US 2012/0208218A1

Patent Application Publication Aug. 16, 2012 Sheet 3 of 7 US 2012/0208218A1

Patent Application Publication Aug. 16, 2012 Sheet 4 of 7 US 2012/0208218A1

FIG. 2A FIG. 2B

FIG. 2C FIG. 2D Patent Application Publication Aug. 16, 2012 Sheet 5 of 7 US 2012/0208218A1

FIG. 3A FIG. 3B

FIG. 3C FIG. 3D Patent Application Publication Aug. 16, 2012 Sheet 6 of 7 US 2012/0208218A1

o S O 7.20 S ? 690 C CD S5 6.60 () As 6.30 o O - is 6.00 CD d

Control Pre-diabetes Diabetes

FIG. 4 Patent Application Publication Aug. 16, 2012 Sheet 7 of 7 US 2012/0208218A1

US 2012/0208218 A1 Aug. 16, 2012

METHODS FOR DETECTING cular changes, and autonomic dysfunction. People with PRE-DABETES AND DABETES USING diabetes are also at increased risk of cardiovascular, periph DIFFERENTAL PROTEIN GLYCOSYLATION eral vascular, and cerebrovascular disease, as well as an increased risk of cancer. Several pathogenic processes are CROSS REFERENCE TO RELATED involved in the development of diabetes, including processes APPLICATIONS that destroy the insulin-secreting beta cells of the pancreas 0001. The present application is a Divisional of and claims with consequent insulin deficiency, and changes in liver and priority to U.S. patent application Ser. No. 12/777,955, filed Smooth muscle cells that result in resistance to insulinuptake. May 11, 2010, entitled “METHODS FOR DETECTING The abnormalities of carbohydrate, fat, and protein metabo PRE-DIABETES AND DIABETES USING DIFFEREN lism are due to deficient action of insulin on target tissues TIAL PROTEIN GLYCOSYLATION” which claims prior resulting from insensitivity to insulin (insulin resistance) or ity to U.S. Provisional Patent Application No. 61/177,130, lack of insulin (loss of beta cell function). filed May 11, 2009, entitled “METHODS FOR DETECTING 0006 Over 18 million people in the United States have PRE-DIABETES AND DIABETES USING DIFFEREN T2DM, and of these, about 5 million do not know they have TIAL PROTEIN GLYCOSYLATION, the disclosures of the disease. These persons, who do not know they have the which are hereby incorporated by reference in their entirety. disease and who do not exhibit the classic symptoms of dia betes, present a major diagnostic and therapeutic challenge. TECHNICAL FIELD Nearly 41 million persons in the United States are at signifi 0002 This relates to the field of diabetes, specifically to cant risk of developing T2DM. These persons are broadly the identification of subjects who have pre-diabetes, diabetes, referred to as “pre-diabetic.” As intervention early in the or diabetic complications. development of diabetes can substantially affect the long term prognosis of the disease, it is beneficial to identify indi BACKGROUND viduals who are pre-diabetic, or those subjects who will become diabetic. 0003 Diabetes mellitus is a metabolic disorder character ized by chronic hyperglycemia with disturbances of carbo hydrate, fat, and protein metabolism that result from defects BRIEF DESCRIPTION OF THE DRAWINGS in insulin secretion, insulin action, or both. Diabetes can present with characteristic symptoms such as thirst, polyuria, 0007 Embodiments will be readily understood by the fol blurring of vision, chronic infections, slow wound healing, lowing detailed description in conjunction with the accom and weight loss. In its most severe forms, ketoacidosis or a panying drawings. Embodiments are illustrated by way of non-ketotic hyperosmolar state may develop and lead to stu example and not by way of limitation in the figures of the por, coma, and, in the absence of effective treatment, death. accompanying drawings. 0004 Diabetes mellitus is subdivided into type 1 diabetes 0008 FIG. 1A is a perspective view of an example of a and type 2 diabetes. Type 1 diabetes (T1DM) results from lateral flow test strip showing the basic components of the auto-immune mediated destruction of the beta cells of the device and their relationship to each other, in accordance with pancreas. Patients with T1DM exhibit little or no insulin various embodiments; secretion as manifested by low or undetectable levels of insu 0009 FIG. 1B is a perspective view of an example of a lin or plasma C-peptide (also known in the art as “soluble lateral flow device showing the test strip enclosed in a hous C-peptide'). Type 2 diabetes (T2DM) is characterized by ing, in accordance with various embodiments; disorders of insulin action and insulin secretion, either of 0010 FIG. 1C shows a perspective view of an example of which may be the predominant feature. T2DM patients can be a lateral flow test strip for the detection of multiple analytes, both insulin deficient and insulin resistant. At least initially, in accordance with various embodiments; and often throughout their lifetime, these individuals do not 0011 FIG. 2 is a series of digital images showing two need supplemental insulin treatment to survive. T2DM dimensional difference gel electrophoresis of serum glyco accounts for 90-95% of all cases of diabetes and can go undiagnosed for many years because the hyperglycemia is proteins from patients with impaired glucose tolerance (IGT. often not severe enough to provoke noticeable symptoms of FIG. 2A), impaired fasting glucose (IFG: FIG. 2B), type 2 diabetes or symptoms are simply not recognized. The major diabetes mellitus (T2DM; FIG.2C), or T2DM (FIG. 2D) fol ity of patients with T2DM are obese, and obesity itself may lowing treatment labeled with Cy5 (patient) or Cy3 (control) cause or aggravate insulin resistance. Many of those who are dyes, in accordance with various embodiments; not obese by traditional weight criteria may have an increased 0012 FIG. 3 is a series of digital images showing two percentage of body fat distributed predominantly in the dimensional difference gel electrophoresis of saliva glyco abdominal region (visceral fat). proteins from patients with impaired glucose tolerance (IGT. 0005. The symptoms of the early stages of diabetes often FIG. 3A), impaired fasting glucose (IFG: FIG. 3B), type 2 are not severe, not recognized, or may be absent. Conse diabetes mellitus (T2DM; FIG. 3C), or type 1 diabetes mel quently, hyperglycemia Sufficient to cause pathological and litus (T1DM: FIG. 3D) labeled with Cy5 (patient) or Cy3 functional changes may be present for a long time, occasion (control) dyes, in accordance with various embodiments; ally up to ten years, before a diagnosis is made, usually by the 0013 FIG. 4 is a graph of mean log-transformed AAL detection of high levels of glucose in urine after overnight binding in urine from control, pre-diabetes, and diabetes Sub fasting during a routine medical work-up. The long-term jects, in accordance with various embodiments; and effects of diabetes include progressive development of com 0014 FIG. 5 is a perspective view of an example of a plications such as retinopathy with potential blindness, neph lateral flow test Strip for diagnosing pre-diabetes or diabetes, ropathy that may lead to renal failure, neuropathy, microvas in accordance with various embodiments. US 2012/0208218 A1 Aug. 16, 2012

DETAILED DESCRIPTION OF DISCLOSED with hepatic insulin resistance and absence of first-phase EMBODIMENTS insulin secretion. These distinctions in location of insulin resistance and extent of beta-cell dysfunction are consistently 0015. In the following detailed description, reference is seen in Native American (Meyer et al., Diabetes Care made to the accompanying drawings which form a part 29:1909-1914, 2006), Mexican-American (Abdul-Ghani et hereof, and in which are shown by way of illustration embodi al., Diabetes 55:1430-1435, 2006), and adult (Laakso et al., ments that may be practiced. It is to be understood that other Diabetologia 51:502-511, 2008) and adolescent (Cali et al., J. embodiments may be utilized and structural or logical Clin. Endocrinol. Metab. 93:1767-1773, 2008) Caucasian changes may be made without departing from the Scope. populations. Therefore, the following detailed description is not to be 0022. However, the use of blood glucose levels as a marker taken in a limiting sense, and the scope of embodiments is of pre-diabetes or frank diabetes has come under recent scru defined by the appended claims and their equivalents. tiny. Specifically, the population-based studies upon which 0016 Various operations may be described as multiple the current diagnostic criteria are based suffered from small discrete operations in turn, in a manner that may be helpful in sample size, inadequate knowledge of the glycemic history of understanding embodiments; however, the order of descrip the participants, and the inherently poor reproducibility of tion should not be construed to imply that these operations are OGTTs (Davidson, Curr. Opin. Endocrin. Diabet. 12:437 order dependent. 443, 2005; Wong et al., Lancet 371:736-743, 2008). These 0017. The description may use perspective-based descrip findings raise serious questions about the adequacy and rel tions such as up/down, back/front, and top/bottom. Such evance of current diagnostic criteria for the diagnosis of descriptions are merely used to facilitate the discussion and T2DM. A separate, but related issue is the consistency of are not intended to restrict the application of disclosed plasma glucose measurements themselves (Gambino, Clin. embodiments. Chem. 53:2040-2041, 2007). Thus, these means of assessing 0018. The terms “coupled and “connected, along with pre-diabetes as well as overt T2DM are fraught with both their derivatives, may be used. It should be understood that technical and patient-Variability issues. these terms are not intended as synonyms for each other. 0023. Although levels of urinary glucose are also unreli Rather, in particular embodiments, “connected may be used able in assessing diabetic status, the evaluation of increased to indicate that two or more elements are in direct physical or levels of protein in urine as well as detection of specific electrical contact with each other. “Coupled may mean that urinary proteins is useful for diagnosis of diabetic nephropa two or more elements are in direct physical or electrical thy and other diseases. More in-depth proteomic analysis of contact. However, “coupled' may also mean that two or more urine is expanding the list of possible urinary biomarkers of elements are not in direct contact with each other, but yet still disease. Among the urine proteins identified by proteomics, cooperate or interact with each other. glycoproteins constitute the largest fraction of the urine pro 0019 For the purposes of the description, a phrase in the teome characterized to date (Adachi et al., Genome Biol. form A/B or in the form 'A and/or B' means (A), (B), or (A 7:R80, 2006; Sun et al., Proteomics 5:4994-5001, 2005; and B). For the purposes of the description, a phrase in the Wang et al., Mol. Cell. Proteomics 5:560-562, 2006). The form “at least one of A, B, and C’ means (A), (B), (C), (A and glycoproteome has been analyzed using Concanavalin B), (A and C), (B and C), or (A, B and C). For the purposes of A-captured glycoproteins from urine samples in healthy Sub the description, a phrase in the form “(A)B' means (B) or jects and those with glomerular disease (Wang et al., Mol. (AB) that is, A is an optional element. Cell. Proteomics 5:560-562, 2006: Wang et al., Biochem. 0020. The description may use the terms "embodiment” or Biophys. Res. Comm. 371:385-390, 3008). Glycoprotein "embodiments.” which may each refer to one or more of the biomarkers from cells and biological fluids such as serum and same or different embodiments. Furthermore, the terms urine have been further characterized by analysis of the “comprising.” “including.” “having.” and the like, as used attached carbohydrate moieties in order to find a more spe with respect to embodiments, are synonymous. cific or easily detectable molecular indicator. Examples include prostate specific antigen (Tabares et al., Glycobiol. I. Introduction 16:132-145, 2006), prion protein species (Pan et al., J. Clin. 0021. The current screening for pre-diabetes includes Microbiol. 43:1118-1126, 2005), fibronectin in rheumatoid measuring blood glucose levels during fasting conditions (to synovial fluid (Przybysz, et al., Glycoconi. J. 24:543-550, detect impaired fasting glucose; IFG) or following a glucose 2007), and alpha-1 acid glycoprotein (A1AG) in amniotic challenge (to determine impaired glucose tolerance; IGT). fluid (Orczyk-Pawilowicz et al., Clin. Chem. Acta 367:86-92, These tests aid in the assessment of potential risk for devel 2006). opment of frank diabetes, such as type 2 diabetes mellitus 0024 Presented herein in various embodiments are direct (T2DM), and facilitates the institution of interventions to lectin assays that may be used to detect changes in the gly slow or prevent disease progression. The definition of IGT as cosylation of biomolecules (glycosylation profile). In a pre-diabetic condition was introduced many years ago to embodiments, changes in a glycosylation profile (such as define disease risk based on oral glucose tolerance tests glycosylation detected by one or more of PHA-E, LEL, DSL, (OGTTs). Subsequently, the classification of IFG was intro ConA, AAL, or SNA) of a sample and/or a glycosylation duced to provide a less complicated and less expensive pattern of one or more specific proteins (such as A1AG and parameter. IFG and IGT are now thought to reflect different A1AT) may be used to identify a subject as a pre-diabetic or aspects of the development of insulin resistance in T2DM, a diabetic or as having a diabetic complication (such as dia with differing underlying pathologies (Abdul-Ghani et al., betic nephropathy). In some examples, the Subjects being Diabetes Care 29:1130-1139, 2006). Specifically, IGT is tested may be diagnosed with IFG and/or IGT (compared to associated with peripheral insulin resistance and loss of first non-diabetic controls) and/or may have newly diagnosed and second-phase insulin secretion, while IFG is associated type-2 diabetes. However, the method also may be used in US 2012/0208218 A1 Aug. 16, 2012 detecting pre-diabetes or diabetes in subjects who have not di-, tri-, and tetraantennary types, with at least twelve glyco been diagnosed with IFG and/or IGT. forms detected in human plasma (Van Dijk et al., Glyconi. J. 0025. Also presented herein in various embodiments are 12:227-233, 1995). assays that may be used to determine the expression level 0055 A1AG sequences are publicly available. For and/or glycosylation pattern of specific proteins (such as example, GenBank Accession number NC 000009.10 A1AG or A1AT). The differences in the glycosylation profile (116125 157 . . . 116128578) discloses an exemplary human of a plurality of biomolecules, and/or the expression or gly Al AG type 1 gene sequence (incorporated by reference as cosylation pattern or amounts of specific proteins between included in GenBank on May 11, 2009). GenBank Accession these conditions may be used in the diagnosis of pre-diabetes number NM 000607.2 and NP 000598.2 disclose exem and/or diabetes. In embodiments, devices that may be used plary human A1AG type 1 cDNA and protein sequences, for measuring the glycosylation profile are also disclosed. respectively (both incorporated by reference as included in GenBank on May 11, 2009). GenBank Accession number II. Terms and Abbreviations NC 000009.10 (116131890 . . . 116135357) discloses an 0026 A1AG: alpha-1 acid glycoprotein exemplary human Al AG type 2 gene sequence (incorporated by reference as included in GenBank on May 11, 2009). 0027 A1AT: alpha-1 antitrypsin GenBank Accession number NM 000608.2 and 0028 ALA: Aleuria aurantia lectin NP 000599.1 disclose exemplary human A1AG type 2 0029 ConA: Concanavalin A cDNA and protein sequences, respectively (both incorpo 0030 DSL: Datura Stramonium lectin rated by reference as included in GenBank on May 11, 2009). 0031 ECL: Erythrina cristagalli lectin One skilled in the art will appreciate that A1AG nucleic acid 0032 ELISA: enzyme-linked immunosorbent assay and protein molecules may vary from those publicly avail 0033 GalNAc: N-acetylgalactosamine able, such as Al AG sequences having one or more substitu 0034 GlcNAc: N-acetylglucosamine tions (for example conservative substitutions), deletions, 0035 GSL-2: Griffonia simplicifolia lectin II insertions, or combinations thereof, while still retaining 0036) HHL: Hippeastrum hybrid lectin A1AG biological activity. In addition, A1AG molecules 0037 IFG: impaired fasting glucose include fragments that retain the desired A1AG biological 0038 IGT: impaired glucose tolerance activity. 0039 LacNAc: N-acetylactosamine 0056 Alpha-1 antitrypsin (A1AT): Also known as alpha-1 0040 LEL: Lycopersicon esculentum lectin proteinase inhibitor or Serpin A1. A 52 kDa serine protease 004.1 LTL: Lotus tetragonolobus lectin inhibitor that is considered the most prominent serpin. The 0.042 MAL: Maackia amurensis lectin I protein was called “antitrypsin' because of its ability to 0043 LEL: Lycopersicon esculentum lectin covalently bind and irreversibly inactivate the enzyme trypsin 0044) LTL: Lotus tetragonolobus lectin in vitro. The term alpha-1 refers to the enzyme’s behavior on protein electrophoresis. There are several “clusters” of pro 0045 NDM: newly diagnosed type-2 diabetes mellitus teins in electrophoresis, the first being albumin, the second 0046 NeuNAc: N-acetylneuraminic acid being the alpha, the third beta and the fourth gamma (immu 0047 OGTT: oral glucose tolerance test noglobulins). The non-albumin proteins are referred to as 0048 PHA-E: Phaseolus vulgaris Agglutinin globulins. The alpha region may be further divided into two 0049 SNA: Sambucus nigra lectin sub-regions, termed “1” and “2. Alpha 1-antitrypsin is the 0050 T2DM: type 2 diabetes main enzyme of the alpha-globulin 1 region. 0051 VVL: Vicia villosa lectin 0057 A1AT sequences are publicly available. For 0.052 Unless otherwise noted, technical terms are used example, GenBank Accession number NC 000014.7 dis according to conventional usage. Definitions of common closes an exemplary human A1 AT gene sequence (93.914451 terms in molecular biology may be found in Benjamin Lewin, . . . 9392.6782) (incorporated by reference as included in Genes V, published by Oxford University Press, 1994 (ISBN GenBank on May 11, 2009). GenBank Accession numbers 0-19-854287-9); Kendrew et al. (eds.). The Encyclopedia of NM 001002235.2, NM 001002236.2, and NM 000295.4 Molecular Biology, published by Blackwell Science Ltd., disclose exemplary human A1AT cDNA sequences (each 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), incorporated by reference as included in GenBank on May Molecular Biology and Biotechnology: a Comprehensive 11, 2009). GenBank Accession numbers NP 001002235.1, Desk Reference, published by VCH Publishers, Inc., 1995 NP 001002236.1, and NP 000286.3 disclose exemplary (ISBN 1-56081-569-8). Definitions and additional informa human A1AT protein sequences (each incorporated by refer tion known to one of skill in the art in immunology may be ence as included in GenBank on May 11, 2009). One skilled found, for example, in Fundamental Immunology, W. E. Paul, in the art will appreciate that A1AT nucleic acid and protein ed., fourth edition, Lippincott-Raven Publishers, 1999. molecules may vary from those publicly available. Such as 0053. In order to facilitate review of the various embodi A1AT sequences having one or more Substitutions (for ments of this disclosure, the following explanations of spe example conservative Substitutions), deletions, insertions, or cific terms are provided: combinations thereof, while still retaining A1AT biological 0054 Alpha-1 acid glycoprotein (A1AG): An approxi activity. In addition, A1AT molecules include fragments that mately 41-43 kDa protein which is one of the major acute retain the desired A1AT biological activity. phase proteins in humans, rats, mice and other species (also 0.058 Analyte: Anatom, molecule, group of molecules or known as orosomucoid). The serum concentration of A1AG compound of natural or synthetic origin (e.g., glycolipids or increases in response to systemic tissue injury, inflammation glycoproteins). An analyte sought to be detected or measured or infection, and is increased in renal complications (such as that is capable of binding specifically to a lectin in some nephropathy). A1AG has five N-linked glycosylation sites of embodiments described herein. Analytes may include, but are US 2012/0208218 A1 Aug. 16, 2012

not limited to antibodies, drugs, hormones, antigens, haptens, with another particular molecule, these two molecules are glycoproteins, glycolipids, carbohydrates, apoproteins, or said to exhibit binding affinity for each other. Binding affinity cofactors. is related to the association constant and dissociation constant 0059 Antibody: A polypeptide ligand comprising at least for a pair of molecules, but it is not critical to the invention a light chain or heavy chain immunoglobulin variable region that these constants be measured or determined. Rather, which specifically binds an epitope of a protein listed in the affinities as used hereinto describe interactions between mol tables below, or a fragment of any of these proteins. The term ecules of the described methods and devices are generally “specifically binds' refers to, with respect to an antigen such apparent affinities (unless otherwise specified) observed in the proteins listed in the tables below, the preferential asso empirical studies, which may be used to compare the relative ciation of an antibody or other ligand, in whole or part, with the protein. A specific binding agent binds Substantially only strength with which one molecule (e.g., an antibody or other to a defined target, such as protein of interest. A minor degree specific binding partner, Such as a lectin) will bind two other of non-specific interaction may occur between a molecule, molecules (e.g., an analyte Such as a glycoprotein). The con Such as a specific binding agent, and a non-target polypeptide. cepts of binding affinity, association constant, and dissocia Specific binding may be distinguished as mediated through tion constant are well known. specific recognition of the antigen. 0065 Biological sample: Any biological sample obtained 0060 A variety of immunoassay formats are appropriate from a plant or animal Subject. As used herein, biological for selecting antibodies specifically immunoreactive with a samples include all clinical samples useful for detection of particular protein. For example, Solid-phase ELISA immu glycosylation profile, protein amount, or glycosylation pat noassays are routinely used to select monoclonal antibodies tern of proteins in Subjects, including, but not limited to, cells, specifically immunoreactive with a protein. See Harlow & tissues, and bodily fluids, such as: blood; derivatives and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor fractions of blood, such as serum; extracted galls; biopsied or Publications, New York (1988), for a description of immu Surgically removed tissue, including tissues that are, for noassay formats and conditions that may be used to determine example, unfixed, frozen, fixed in formalin and/or embedded specific immunoreactivity. in paraffin, tears; mucus; saliva; milk; skin scrapes; Surface 0061 Antibodies may include a heavy chain and a light washings; urine; sputum; Sweat; semen; vaginal Secretion; chain, each of which has a variable region, termed the variable fluid from ulcers and/or other surface eruptions, blisters, heavy (VH) region and the variable light (VL) region. abscesses, and/or extracts of tissues; cells or organs; cere Together, the VH region and the VL region are responsible for brospinal fluid; prostate fluid; pus; or bone marrow aspirates. binding the antigen recognized by the antibody. This includes The biological sample may also be a laboratory research intact immunoglobulins and the variants and portions of them sample such as a cell culture Supernatant. In particular well known in the art, such as Fab' fragments, F(ab)2 frag examples, the sample is urine, saliva, or serum. The sample is ments, single chain Fv proteins (“scFv'), and disulfide stabi collected or obtained using methods well known to those lized FV proteins (“dsFv'). A schv protein is a fusion protein skilled in the art. in which a light chain variable region of an immunoglobulin 006.6 Biomolecule: A molecule that is present in a bio and a heavy chain variable region of an immunoglobulin are logical sample including, but not limited to, proteins (such as bound by a linker, while in dsEvs, the chains have been polypeptides, proteins, or fragments thereof), lipids, and mutated to introduce a disulfide bond to stabilize the associa nucleic acids. The term “biomolecule' is specifically tion of the chains. The term also includes recombinant forms intended to cover naturally occurring biomolecules, as well as Such as chimericantibodies (for example, humanized murine those which are recombinantly or synthetically produced. It antibodies), heteroconjugate antibodies (such as, bispecific should be noted that the term “biomolecule' includes modi antibodies). See also, Pierce Catalog and Handbook, 1994 fied forms of the biomolecules, such as glycosylated forms 1995 (Pierce Chemical Co., Rockford, Ill.); Kuby. Immunol (for example, glycosylated proteins or peptides or glycosy ogy, 3rd Ed., W.H. Freeman & Co., New York, 1997. lated lipids). 0062. A "monoclonal antibody' is an antibody produced 0067 Capture reagent: An unlabeled specific binding by a single clone of B-lymphocytes or by a cell into which the partner that is specific for (i) an analyte, as in a sandwich light and heavy chain genes of a single antibody have been assay, or (ii) a detector reagent or an analyte, as in a competi transfected. Monoclonal antibodies are produced by methods tive assay, or for (iii) an ancillary specific binding partner, known to those of skill in the art, for instance by making which itself is specific for the analyte, as in an indirect assay. hybrid antibody-forming cells from a fusion of myeloma cells As used herein, an “ancillary specific binding partner is a with immune spleen cells. These fused cells and their progeny specific binding partner that binds to the specific binding are termed “hybridomas.” Monoclonal antibodies include partner of an analyte. For example, an ancillary specific bind humanized monoclonal antibodies. ing partner may include an antibody specific for another 0063 Antigen: A chemical or biochemical compound, antibody, for example, goat anti-human antibody. A "capture composition, structure, determinant, protein, glycoprotein or area' is a region of a lateral flow device where the capture portion thereofthat may stimulate the production of antibod reagent is immobilized. A lateral flow device may have more ies or a T-cell response in an animal, including compositions than one capture area, for example, a “primary capture area.” that are injected or absorbed into an animal. An antigen reacts a 'secondary capture area, and so on. Often a different cap with the products of specific humoral or cellular immunity, ture reagent will be immobilized in the primary, secondary, or including those induced by heterologous immunogens. The other capture areas. Multiple capture areas may have any term “antigen' includes all related antigenic epitopes. orientation with respect to each other on the lateral flow 0064 Binding affinity: A term that refers to the strength of Substrate; for example, a primary capture area may be distal or binding of one molecule to another at a site on the molecule. proximal to a secondary (or other) capture area and vice versa. If a particular molecule will bind to or specifically associate Alternatively, a primary capture area and a secondary (or US 2012/0208218 A1 Aug. 16, 2012 other) capture area may be oriented perpendicularly to each opathy, diabetic neuropathy, and diabetic cardiomyopathy. other Such that the two (or more) capture areas form a cross or Macrovascular complications lead to cardiovascular disease a plus sign or other symbol. and include coronary artery disease, stroke, peripheral vas 0068 Contacting: “Contacting includes in solution and cular disease, and diabetic myonecrosis. Additional chronic Solid phase, for example contacting a salivary sample, urinary diabetic complications include diabetic foot, which results sample or protein with a test agent. In one example, contact from a combination of diabetic neuropathy and peripheral ing includes contacting a sample with a lectin, such as those vascular disease, and diabetic encephalopathy. In a particular listed in Table 1 below. In another example, contacting example, the methods disclosed herein include diagnosis of includes contacting a sample with an antibody, for example one or more diabetic complication, Such as microvascular contacting a sample that contains a protein of interest Such as complications, for example, diabetic nephropathy. alpha-1 acid glycoprotein or alpha-1 antitrypsin. 0075 Glycosylation: Covalent modification of a biomol 0069 Detecting or Detection: Refers to quantitatively or ecule (such as a protein or lipid) with one or more oligosac quantitatively determining the presence of the analyte(s) charide chains. Proteins having at least one oligosaccharide under investigation, such as a glycoprotein. modification are referred to as 'glycoproteins' or 'glycosy 0070 Detector reagent (or Detection reagent): A specific lated proteins. In the case of proteins, glycosylation is usu binding partner that is conjugated to a label. ally N-linked or O-linked. N-linked glycosylation refers to 0071. Diabetes mellitus: A disease caused by a relative or linkage of an oligosaccharide to the side chainamino group of absolute lack of insulin leading to uncontrolled carbohydrate an asparagine residue in a protein. O-linked glycosylation metabolism, commonly simplified to “diabetes, though dia refers to linkage of an oligosaccharide to the hydroxyl side betes mellitus should not be confused with diabetes insipidus. chain of a serine, threonine, or hydroxylysine amino acid in a As used herein, "diabetes’ refers to diabetes mellitus, unless protein. otherwise indicated. A "diabetic condition' includes pre-dia 0076. The oligosaccharide chains of glycoproteins are betes and diabetes. Type 1 diabetes (sometimes referred to as enormously varied, due to the combination of various Sugars “insulin-dependent diabetes’ or “juvenile-onset diabetes') is (for example, N-acetylglucosamine, N-acetylgalactosamine, an auto-immune disease characterized by destruction of the N-acetylactosamine, mannose, galactose, glucose, N-acetyl pancreatic B cells that leads to a total or near total lack of neuraminic acid, or fucose) and the presence of branched insulin. In type 2 diabetes (T2DM; sometimes referred to as structures (such as biantennary, triantennary, or tetra-anten “non-insulin-dependent diabetes’ or “adult-onset diabetes’), nary structures). the body does not respond to insulin, though it is present. As 0077. Glycosylation profile: A representation of the gly used herein, the term “metabolic condition' is used to refer to cosylation (such as the amount or type of glycosylation) of a type 1 diabetes, type 2 diabetes, pre-diabetes, and diabetes plurality of biomolecules (such as a plurality of glycoproteins complications. or glycolipids) in a biological sample, Such as urine or saliva. 0072 Symptoms of diabetes include: excessive thirst The glycosylation profile provides information on the amount (polydipsia); frequent urination (polyuria); extreme hunger of one or more type of carbohydrate group (for example, or constant eating (polyphagia); unexplained weight loss; N-acetylglucosamine, N-acetyl galactosamine, galactose, presence of glucose in the urine (glycosuria); tiredness or neuraminic acid, fructose, mannose, fucose, N-acetyllac fatigue; changes in vision; numbness or tingling in the tosamine) or branch structure (such as bi-, tri-, or tetra-anten extremities (hands, feet); slow-healing wounds or Sores; and nary) present on the plurality of biomolecules present in the abnormally high frequency of infection. Diabetes may be sample. The glycosylation profile of a plurality of biomol clinically diagnosed by a fasting plasma glucose (FPG) con ecules in a sample may be determined by detecting of binding centration of greater than or equal to 7.0 mmol/L (126 of biomolecules in the sample to one or more lectins. In some mg/dL), or a plasma glucose concentration of greater than or examples, the glycosylation profile of a sample from a subject equal to 11.1 mmol/L (200 mg/dL) at about two hours after an is compared to the glycosylation profile of a reference in order oral glucose tolerance test (OGTT) with a 75 g load. A more to determine whether the subject has pre-diabetes, diabetes, detailed description of diabetes may be found in Cecil Text or a diabetic complication. book of Medicine, J. B. Wyngaarden, et al., eds. (W.B. Saun 0078 Glycosylation pattern: The carbohydrate groups ders Co., Philadelphia, 1992, 19" ed.). attached to a particular biomolecule (such as a particular 0073. The methods disclosed herein provide a means of glycoprotein or glycolipid). Such as the number, structure, identifyings Subject who has diabetes or pre-diabetes, includ monosaccharide sequence, or location of the individual Sug ing both type 1 and type 2 diabetes. A “non-diabetic' or ars on the biomolecule in a biological sample, Such as urine or “normal subject does not have any form of diabetes, such as saliva. The glycosylation pattern of a biomolecule may be type 1 diabetes, type 2 diabetes, or pre-diabetes. determined by detecting binding of the biomolecule to one or 0074 Diabetic complication: Pathologies associated with more lectins (such as by lectin-ELISA). In particular diabetes that are secondary to uncontrolled carbohydrate examples, the glycosylation pattern of a glycoprotein (such as metabolism of diabetes. Acute complications (such as A1AG or A1AT) in a sample from a subject is determined. In hypoglycemia, ketoacidosis, or nonketotic hyperosmolar Some examples, the glycosylation pattern of A1AG and/or coma) may occur if the blood Sugar is not adequately con A1AT in a sample from a subject is compared to the glyco trolled. Chronic elevation of blood glucose level leads to sylation pattern of A1AG and/or A1AT in a reference in order damage of blood vessels (angiopathy) and chronic complica to determine whether the subject has pre-diabetes, diabetes, tions. In diabetes, the resulting chronic complications are or a diabetic complication. grouped under “microvascular complications” (due to dam 0079 Immunoassay: A biochemical test that measures the age to Small blood vessels) and "macrovascular complica presence or concentration of a Substance in a sample, Such as tions” (due to damage to the arteries). Microvascular diabetic a biological sample, using the reaction of an antibody to its complications include diabetic nephropathy, diabetic retin cognate antigen, for example the specific binding of an anti US 2012/0208218 A1 Aug. 16, 2012 body to a protein. The presence of antigen and/or the amount examples an antibody that specifically binds the lectin is of antigen present may be measured. For measuring proteins, labeled with a detectable marker, such as horseradish peroxi for each antigen the presence and amount (abundance) of the dase. Methods for labeling and guidance in the choice of protein may be determined or measured. The assay may be labels appropriate for various purposes are discussed for competitive or non-competitive. example in Sambrook et al. (Molecular Cloning: A Labora 0080 Measuring the quantity of antigen (such as A1AG or tory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et A1AT) may be achieved by a variety of methods. One of the al. (In Current Protocols in Molecular Biology, John Wiley & most common is to label either the antigen orantibody with a Sons, New York, 1998), Harlow & Lane (Antibodies, A Labo detectable label. Specific, non-limiting examples of labels ratory Manual, Cold Spring Harbor Publications, New York, include fluorescent tags, enzymatic linkages, and radioactive isotopes (for example “C. P. ''I, and H isotopes and the 1988). like). In some examples, an antibody that specifically binds 0084. Lateral flow device: A device that absorbs or one of an antigen of interest is labeled. Methods for labeling adsorbs a liquid sample, routes that liquid sample to a detec and guidance in the choice of labels appropriate for various tion Zone, and uses antibody- or lectin-based detection meth purposes are discussed for example in Sambrook et al. (Mo ods to generate a visible signal in response to the presence or lecular Cloning: A Laboratory Manual, Cold Spring Harbor, absence of a specific antigen (such as a protein or glycopro N.Y., 1989) Ausubeletal. (In Current Protocols in Molecular tein) or lectin-binding biomolecule (such as a glycoprotein or Biology, John Wiley & Sons, New York, 1998), and Harlow & glycolipid). The device may be a test strip used in lateral flow Lane, (Antibodies, A Laboratory Manual, Cold Spring Har chromatography, in which a test sample fluid, Suspected of bor Publications, New York, 1988)). containing an analyte, flows (for example by capillary action) 0081) Isolated: An isolated biological component (such as through the strip (which is frequently made of bibulous mate a nucleic acid, peptide or protein) has been Substantially rials such as paper, nitrocellulose, and cellulose). The test separated, produced apart from, or purified away from other fluid and any Suspended analyte may flow along the strip to a biological components in the cell of the organism in which the detection Zone in which the analyte (if present) interacts with component naturally occurs, for example the separation of a a detection agent to indicate a presence, absence and/or quan peptide from a sample, such as saliva, urine, serum or blood. tity of the analyte. Peptides and proteins that have been isolated include proteins I0085 Numerous lateral flow analytical devices have been purified by standard purification methods, Such as chroma disclosed, and include those shown in U.S. Pat. Nos. 4.313, tography, for example high performance liquid chromatogra 734; 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4.806,311: phy (HPLC) and the like. The term also embraces peptides, 4,806,312; 4,861,711; 4,855,240; 4,857,453: 4,943,522; and proteins prepared by recombinant expression in a host 4,945,042; 4,496,654; 5,001,049; 5,075,078; 5,126,241: cell as well as chemically synthesized peptide and nucleic 5,451,504; 5,424, 193; 5,712,172; 6.258,548; 6,555,390; acids. It is understood that the term "isolated' does not imply 6,699,722; and 6,368,876; EP 0810436; and WO92/12428; that the biological component is free of trace contamination, WO 94/01775; WO95/16207; and WO 97/06439, each of and may include molecules that are at least 50% isolated, such which is incorporated by reference. as at least 50%, 55%, 60%. 65%, 70%, 75%, 80%, 85%, 90%, I0086. Many lateral flow devices are one-step lateral flow 95%, 98%, 99%, or even 100% isolated. assays in which a biological fluid is placed in a sample area on 0082 Label: Any molecule or composition bound to an abibulous strip (though, non-bibulous materials may be used, analyte, analyte analog, detector reagent, or binding partner and rendered bibulous by applying a surfactant to the mate that is detectable by spectroscopic, photochemical, biochemi rial), and allowed to migrate along the strip until the liquid cal, immunochemical, electrical, optical or chemical means. comes into contact with a specific binding partner (Such as a Examples of labels, including enzymes, colloidal gold par lectin or antibody) that interacts with an analyte (such as a ticles, colored latex particles, have been disclosed (U.S. Pat. glycoprotein, glycolipid, or antigen) in the liquid. Once the Nos. 4,275,149; 4.313,734; 4,373,932; and 4,954,452, each analyte interacts with the binding partner, a signal (such as a incorporated by reference herein). Additional examples of fluorescent or otherwise visible dye) indicates that the inter useful labels include, without limitation, radioactive isotopes, action has occurred. Multiple discrete binding partners may co-factors, ligands, chemiluminescent or fluorescent agents, be placed on the strip (for example in parallel lines) to detect protein-adsorbed silver particles, protein-adsorbed iron par multiple analytes in the liquid. The test strips may also incor ticles, protein-adsorbed copper particles, protein-adsorbed porate control indicators, which provide a signal that the test Selenium particles, protein-adsorbed Sulfur particles, protein has adequately been performed, even if a positive signal indi adsorbed tellurium particles, protein-adsorbed carbon par cating the presence (or absence) of an analyte is not seen on ticles, and protein-coupled dye sacs. The attachment of a the strip. compound (e.g., a detector reagent) to a label may be through I0087 Lectin: A carbohydrate-binding protein, some of covalent bonds, adsorption processes, hydrophobic and/or which are specific for one or more particular carbohydrate electrostatic bonds, as in chelates and the like, or combina moieties. Most known lectins are multimeric, consisting of tions of these bonds and interactions and/or may involve a non-covalently associated Subunits. A lectin may contain two linking group. or more of the same subunit, such as Concanavalin A (ConA), 0083. In some examples, a label is conjugated directly or or different subunits, such as Phaseolus vulgaris agglutinin indirectly to another molecule. Such as an antibody or a pro (PHA-E). In particular examples, lectins include Aleuria tein, to facilitate detection of that molecule. Specific, non aurantia lectin (AAL), Concanavalin A (Con A), Datura limiting examples of labels include fluorescent tags, enzy stramonium lectin (DSL), Erythrina Cristagalli lectin (ECL), matic linkages, and radioactive isotopes (for example ''C, Griffonia simplicifolia lectin II (GSL-2), Hippeastrum hybrid 'P, 'I, H isotopes and the like). In some examples a lectin lectin (HHL), Lycopersicon esculentum lectin (LEL), Lotus is labeled with a detectable marker, such as biotin. In some tetragonolobus lectin (LTL), Maackia amurensis lectin I US 2012/0208218 A1 Aug. 16, 2012

(MAL), Phaseolus vulgaris Agglutinin (PHA-E), Sambucus than or equal to 200 mg/dl indicates that a Subject has frank nigra lectin (SNA), and Vicia villosa lectin (VVL). diabetes, and an OGTT of less than 140 mg/dl indicates that 0088 Linking group: A chemical arm between two com a subject is normal (healthy) and does not have pre-diabetes pounds, for instance a compound and a label (e.g., an analyte, or diabetes. Such as a glycoprotein or a glycolipid, and a label). To accom 0093 Generally, impaired fasting glucose (IFG) may also plish the requisite chemical structure, each of the reactants be used to identify a Subject as pre-diabetic. Fasting plasma must contain a reactive group. Representative combinations glucose (FPG) of greater than 100 mg/dL and less than 126 of Such groups are amino with carboxyl to form amide link mg/dL (5.6-6.9 mM) indicates that a subject has IFG and has ages; carboxy with hydroxy to form ester linkages or amino pre-diabetes. FPG of greater than or equal to 126 mg/dl indi with alkyl halides to form alkylamino linkages; thiols with cates that a subject has frank diabetes, and an FPG of equal to thiols to form disulfides; or thiols with maleimides or alkyl or less than 100 mg/dl indices that subject is normal (healthy) halides to form thioethers. Hydroxyl, carboxyl, amino and and does not have pre-diabetes or diabetes. other functionalities, where not present in the native com 0094 Sample application area: An area where a fluid pound, may be introduced by known methods. sample is introduced to a immunochromatographic test strip, 0089. Likewise, a wide variety of linking groups may be Such as an immunochromatographic test strip present in a employed. The structure of the linkage should be a stable lateral flow device. In one example, the sample may be intro covalent linkage formed to attach two compounds to each duced to the sample application area by external application, other (e.g., the label to the analyte). In some cases the linking as with a dropper or other applicator. In another example, the group may be designed to be either hydrophilic or hydropho sample application area may be directly immersed in the bic in order to enhance the desired binding characteristics, for sample, Such as when a test strip is dipped into a container instance of the modified ligand and its cognate receptor. The holding a sample. In yet another example, the sample may be covalent linkages should be stable relative to the solution poured or expressed onto the sample application area. conditions to which linked compounds are Subjected. 0.095 Solid support (or substrate): Any material which is Examples of linking groups will be from 1-20 carbons and insoluble, or may be made insoluble by a Subsequent reaction. 0-10 heteroatoms (NH, O, S) and may be branched or straight Numerous and varied solid Supports are known to those in the chain. Without limiting the foregoing, it should be obvious art and include, without limitation, nitrocellulose, the walls of that only combinations of atoms that are chemically compat wells of a reaction tray, multi-well plates, test tubes, polysty ible comprise the linking group. For example, amide, ester, rene beads, magnetic beads, membranes, and microparticles thioether, thiolester, keto, hydroxyl, carboxyl, ether groups in (such as latex particles). Any suitable porous material with combinations with carbon-carbon bonds are particular Sufficient porosity to allow access by detector reagents and a examples of chemically compatible linking groups. Suitable Surface affinity to immobilize capture reagents (e.g., 0090 Polypeptide: A polymer in which the monomers are lectins or antibodies) is contemplated by this term. For amino acid residues which are joined together through amide example, the porous structure of nitrocellulose has excellent bonds. When the amino acids are alpha-amino acids, either absorption and adsorption qualities for a wide variety of the L-optical isomer or the D-optical isomer may be used, the reagents, for instance, capture reagents. Nylon possesses L-isomers being preferred. The terms “polypeptide' or “pro similar characteristics and is also Suitable. Microporous tein' or “peptide' as used herein are intended to encompass structures are useful, as are materials with gel structure in the any amino acid sequence and include modified sequences hydrated state. such as glycoproteins. The term “polypeptide' or “protein’ or 0096. Further examples of useful solid supports include: "peptide' is specifically intended to cover naturally occurring natural polymeric carbohydrates and their synthetically proteins, as well as those which are produced by recombinant modified, cross-linked or substituted derivatives, such as or synthetic methods. agar, agarose, cross-linked alginic acid, Substituted and cross 0091 Operable or contiguous contact: Two solid compo linked guar gums, cellulose esters, especially with nitric acid nents are in operable contact when they are in contact, either and carboxylic acids, mixed cellulose esters, and cellulose directly or indirectly, in Such a manner that an aqueous liquid ethers; natural polymers containing nitrogen, Such as proteins may flow from one of the two components to the other sub and derivatives, including cross-linked or modified gelatins: stantially uninterruptedly, by capillarity or otherwise. Direct natural hydrocarbon polymers, such as latex and rubber, Syn or contiguous contact means that the two elements are in thetic polymers which may be prepared with suitably porous physical contact, such as edge-to-edge or front-to-back. structures, such as vinyl polymers, including polyethylene, When two components are in direct contact, they may overlap polypropylene, polystyrene, polyvinylchloride, polyvinylac with an overlap of about 0.5 mm to about 3 mm. However, the etate and its partially hydrolyzed derivatives, polyacryla components may be placed with abutting edges. “Indirect mides, polymethacrylates, copolymers and terpolymers of contact” means that the two elements are not in physical the above polycondensates, such as polyesters, polyamides, contact, but are bridged by one or more conductors. Operable and other polymers, such as polyurethanes or polyepoxides; contact may also be referred to as “fluid transmitting or porous inorganic materials such as Sulfates or carbonates of “fluid continuous’ contact. alkaline earth metals and magnesium, including barium Sul 0092 Pre-diabetes: A condition identified in a subject by fate, calcium sulfate, calcium carbonate, silicates of alkali impaired glucose tolerance, alone or in combination with and alkaline earth metals, aluminum and magnesium; and impaired fasting glucose regulation. An oral glucose toler aluminum or silicon oxides or hydrates, such as clays, alu ance test (OGTT) may be used to determine if a subject has mina, talc, kaolin, Zeolite, silica gel, or glass (these materials impaired glucose tolerance (IGT). An OGTT two-hour may be used as filters with the above polymeric materials); plasma glucose of greater than or equal to 140 mg/dL and less and mixtures or copolymers of the above classes, such as graft than 200 mg/dL (7.8-11.0 mM) is considered to be IGT, and copolymers obtained by initializing polymerization of Syn indicates that a subject has pre-diabetes. An OGTT of greater thetic polymers on a pre-existing natural polymer. US 2012/0208218 A1 Aug. 16, 2012

0097. It is contemplated that porous solid supports, such as nated assay conditions, the lectins or antibodies bind to a nitrocellulose, described herein are preferably in the form of particular analyte. Such as proteins, glycoproteins and/or gly sheets or strips. The thickness of Such sheets or strips may colipids, and do not bind in a significant amount to other vary within wide limits, for example, from about 0.01 to 0.5 analytes present in the sample. A variety of assay formats may mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, be used to select lectins or antibodies specifically reactive from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or with a particular analyte, such as a glycoprotein or glycolipid from about 0.11 to 0.15 mm. The pore size of such sheets or or a particular protein. For example, solid-phase ELISA strips may similarly vary within wide limits, for example immunoassays are routinely used to select monoclonal anti from about 0.025 to 15 microns, or more specifically from bodies specifically immunoreactive with a protein. See Har 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 low and Lane, Antibodies, A Laboratory Manual, CSHP, New rate of a Solid Support, where applicable, may also vary within York (1988), for a description of immunoassay formats and widelimits, for example from about 12.5 to 90 sec/cm (i.e., 50 conditions that may be used to determine specific immunore to 300 sec/4 cm), about 22.5 to 62.5 sec/cm (i.e., 90 to 250 activity. sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250 sec/4 cm), 0104 Subject: Living multi-cellular vertebrate organisms, about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), or about a category that includes human and non-human mammals 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specific (such as laboratory or veterinary Subjects). embodiments of devices described herein, the flow rate is 0105 Substantially the same: An amount that is not sig about 62.5 sec/cm (i.e., 250 sec/4 cm). In other specific nificantly different from a reference or control. This may be embodiments of devices described herein, the flow rate is measure quantitatively, Such as by using statistical methods, about 37.5 sec/cm (i.e., 150 sec/4 cm). for example a Student's T-test or ANOVA. For example, a 0098. The surface of a solid support may be activated by glycosylation profile of a sample from a subject is substan chemical processes that cause covalent linkage of an agent tially the same as a glycosylation profile of a sample from a (e.g., a capture reagent) to the Support. However, any other control Subject when the amount of glycosylation detected by Suitable method may be used for immobilizing an agent (e.g., one or more lectins is not different between the two samples. a capture reagent) to a solid Support including, without limi 0106 Unless otherwise explained, all technical and scien tation, ionic interactions, hydrophobic interactions, covalent tific terms used herein have the same meaning as commonly interactions and the like. The particular forces that result in understood by one of ordinary skill in the art to which this immobilization of an agent on a solid phase are not important disclosure belongs. The singular terms “a,” “an,” and “the for the methods and devices described herein. include plural referents unless context clearly indicates oth 0099. A solid phase may be chosen for its intrinsic ability erwise. Similarly, the word 'or' is intended to include “and” to attract and immobilize an agent, such as a capture reagent. unless the context clearly indicates otherwise. It is further to Alternatively, the Solid phase may possess a factor that has the be understood that all base sizes or amino acid sizes, and all ability to attract and immobilize an agent, Such as a capture molecular weight or molecular mass values, given for nucleic reagent. The factor may include a charged Substance that is acids or polypeptides are approximate, and are provided for oppositely charged with respect to, for example, the capture description. Although methods and materials similar or reagent itself or to a charged substance conjugated to the equivalent to those described herein may be used in the prac capture reagent. In another embodiment, a specific binding tice or testing of this disclosure, Suitable methods and mate member may be immobilized upon the Solid phase to immo rials are described below. The term “comprises' means bilize its binding partner (e.g., a capture reagent). In this “includes. All publications, patent applications, patents, and example, therefore, the specific binding member enables the other references mentioned herein are incorporated by refer indirect binding of the capture reagent to a solid phase mate ence in their entirety. All GenBank Accession Nos. men rial. tioned herein are incorporated by reference in their entirety. 0100 Except as otherwise physically constrained, a solid In case of conflict, the present specification, including expla Support may be used in any Suitable shapes, such as films, nations of terms, will control. In addition, the materials, sheets, strips, or plates, or it may be coated onto or bonded or methods, and examples are illustrative only and not intended laminated to appropriate inert carriers, such as paper, glass, to be limiting. plastic films, or fabrics. III. Methods for Identifying a Subject with Pre-Diabetes or 0101. A “lateral flow substrate' is any solid support or Diabetes substrate that is useful in a lateral flow device. 0107. In various embodiments, methods are disclosed 0102 Specific binding partner (or binding partner): A herein that may be of use to determine whether a subject has member of a pair of molecules that interact by means of a diabetic condition, for instance pre-diabetes or diabetes. In specific, noncovalent interactions that depend on the three Some embodiments, these methods may utilize a biological dimensional structures of the molecules involved. Typical sample (such as urine, Saliva, blood, serum, amniotic fluid, or pairs of specific binding partners include antigen/antibody, tears, for example, saliva, urine, or serum), for the detection carbohydrateflectin, hapten/antibody, hormone/receptor, of a glycosylation profile of a plurality of biomolecules (such nucleic acid strand/complementary nucleic acid strand, Sub as glycoproteins or glycolipids) in the sample. In other strate/enzyme, inhibitor/enzyme, biotin/(strept)avidin, and embodiments, the glycosylation pattern of one or more spe virus/cellular receptor. cific proteins (such as A1AG or A1AT) may also be used to 0103) The phrase “specifically binds to an analyte' or determine if a subject has a diabetic condition, Such as pre “specifically immunoreactive with.” refers to a binding reac diabetes or diabetes. In some examples, the methods also may tion which is determinative of the presence of the analyte in include utilizing a biological sample (for example urine or the presence of a heterogeneous population of molecules Such saliva) for determining the amount of one or more proteins, as proteins and other biologic molecules. Thus, under desig including, but not limited to, A1AG or A1AT. In additional US 2012/0208218 A1 Aug. 16, 2012 examples, the methods also may include determining the amount or type of glycosylation) relative to the reference, the glycosylation pattern of A1AG or A1AT. subject is determined not to have pre-diabetes or diabetes, respectively. A. Glycosylation Profile 0112. In particular examples, the method may include 0108. In some embodiments, the methods disclosed herein detecting an increase, such as at least about a 2, 3, 4, 5, 6, 7, may include determining a glycosylation profile (such as the 8, 9, 10, 12, 15, or 20-fold increase (for example about a amount or type of glycosylation of a plurality of biomol 2-fold to 20-fold, 3-fold to 15-fold, or 5-fold to 10-fold ecules) of a biological sample (such as a biological fluid, for increase), in at least one informative parameter of the glyco example, urine or saliva). In embodiments, a change in the Sylation profile, such as the amount of one or more type of glycosylation profile of a sample may reflect a change in the glycosylation (such as N-glycosylation, for example, one or amount or type of glycosylation (such as N-glycosylation) of more carbohydrate group, such as N-acetylglucosamine, a particular biomolecule(s) as compared to the amount or type N-acetylgalactosamine, N-acetylactosamine, N-acetyl of the particular biomolecule(s) in a reference standard, Such neuraminic acid, mannose, fucose, or galactose), or the as a reference sample or quantity. A change in the glycosyla amount of one or more oligosaccharide structures (such as a tion profile may also reflect the presence of glycosylation bi-antennary, tri-antennary, or tetra-antennary structure) (such as N-glycosylation) of a particular biomolecule(s) that present in a sample. An "informative parameter refers to a is not present on the particular biomolecule(s) in a reference, measure (such as a quantity of biomolecule binding to a or the absence of glycosylation (such as N-glycosylation) of particular lectin) that detects a type of glycosylation associ a particular biomolecule(s) that is present on the particular ated with pre-diabetes or diabetes. biomolecule(s) in a reference. Thus, in various embodiments, 0113. In particular examples, the amount of glycosylation the glycosylation profile may not provide information about may be detected by determining an amount of a sample bind modification of any one specific biomolecule in the sample, ing to a lectin (such as PHA-E, LEL, DSL, ConA, AAL, SNA, but may provide information about the amount or type of or MAL). In some examples, the method may include detect glycosylation (such as N-glycosylation) present on any bio ing an increase in biomolecule glycosylation recognized by molecule present in the sample or on a population of biomol the lectin PHA-E (such as at least about a 2, 3, 4, or 5-fold ecules in the sample. increase, for example about a 2-fold or 3-fold increase). Such 0109. In some embodiments, the glycosylation profile of a as lactosamine glycosylation (for example, Gal-61.4 sample is determined by measuring the binding of biomol GlcNAc), in a subject with pre-diabetes or diabetes. In addi ecules in a biological sampleto one or more lectins. In various tional examples, the method may include detecting an embodiments, lectins may bind specifically to particular car increase in biomolecule glycosylation recognized by the lec bohydrates or oligosaccharide structures, such as those tin LEL (such as at least about a 2, 3, 4, or 5-fold increase, for present in glycoproteins or glycolipids (see, e.g., Section V). example about a 2-fold or 3-fold increase), such as trimers or In some examples, a biological sample (such as urine, Saliva, tetramers of GlcNAc, in a subject with pre-diabetes or diabe or serum) which contains a plurality of biomolecules may be tes. In additional examples, the method may include detecting contacted with one or more lectins, and binding of biomol an increase in biomolecule glycosylation recognized by the ecule(s) to the lectin(s) is detected. In various embodiments, lectin AAL (Such as at least about a 2, 3, 4, or 5-fold increase, the amount of biomolecule(s) binding to a lectin having a for example about a 2-fold or 3-fold increase), such as fucose, particular specificity indicates the amount of that particular in a subject with pre-diabetes or diabetes. type of glycosylation present on the plurality of biomolecule 0114. In further examples, the method may include detect (s) in the sample. ing an increase in biomolecule glycosylation recognized by 0110. In some embodiments, the method includes com the lectin DSL (such as at least about a 2, 3, 4, or 5-fold paring a glycosylation profile of a test sample (such as urine, increase, for example about a 2-fold or 3-fold increase) in a saliva, or serum) from a Subject of interest with a glycosyla subject with pre-diabetes or diabetes. In additional examples, tion profile of a reference standard. Such as a reference sample the method may include detecting an increase in biomolecule or quantity. In one embodiment, the method may determine glycosylation recognized by the lectin ConA (Such as at least whether the subject has pre-diabetes or diabetes. In embodi about a 2-fold to 20-fold increase, for example about a 2-fold, ments, if the reference is a normal reference and the glyco 5-fold, 10-fold, 15-fold, or 20-fold increase) in a subject with Sylation profile of the test sample is Substantially the same as pre-diabetes or diabetes. In still further examples, the method the glycosylation profile of the reference (for example, the may include detecting an increase in biomolecule glycosyla amount or type of glycosylation is Substantially the same), the tion recognized by the lectin SNA (such as at least about a subject is determined not to have pre-diabetes or diabetes, 2-fold to 15-fold increase, for example about a 2-fold, 5-fold, respectively. However, in embodiments, if the glycosylation 10-fold, or 15-fold increase) in a subject with pre-diabetes or profile of the test sample is changed relative to the glycosy diabetes. In some examples, a glycosylation profile may lation profile of the reference (for example, the amount or include the amount of sample binding to PHA-E, LEL, AAL, type of glycosylation is increased), the Subject is determined DSL, ConA, SNA, MAL, or a combination of two or more to have pre-diabetes or diabetes, respectively. thereof. 0111. In another embodiment, if the reference is a pre 0.115. In still further examples, the amount of the increase diabetes or diabetes reference and the glycosylation profile of of glycosylation detected by sample binding to a lectin (Such the test sample is Substantially the same as the reference as ConA or SNA) may be useful to differentiate whether a sample (for example the amount or type of glycosylation is Subject has pre-diabetes or diabetes. For example, an increase essentially the same, such as not significantly different), then in sample binding to ConA of about 2-fold to 8-fold (such as the subject may be determined to have pre-diabetes or diabe about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold) tes, respectively. In embodiments, of the glycosylation profile may indicate that the Subject has pre-diabetes, while an of the test sample is changed (for example, a decrease in the increase in sample binding to ConA of about 10-fold to US 2012/0208218 A1 Aug. 16, 2012

20-fold (such as about 10-fold, 12-fold, 15-fold, 18-fold, or determined not to have pre-diabetes or diabetes. However, if 20-fold) may indicate that the subject has diabetes. In another the amount of one or more protein of the test sample is example, an increase in sample binding to SNA of about changed relative to the amount of said protein of the refer 2-fold to about 5-fold (such as about 2-fold, 3-fold, 4-fold, or ence, the subject may be determined to have pre-diabetes or 5-fold) may indicate that the subject has pre-diabetes, while diabetes. an increase in sample binding to SNA of about 8-fold to about I0120 In another embodiment, if the reference is a pre 15-fold (such as about 8-fold, 9-fold, 10-fold, 12-fold, or diabetes or diabetes reference, and the amount of one or more 15-fold) may indicate that the subject has diabetes. protein in the test sample is Substantially the same as the 0116. In an additional embodiment, the method may amount of said protein in the reference, then the Subject may include detecting an increase in a ratio of sample binding to be determined to have pre-diabetes or diabetes, respectively. two lectins, such as a test lectin and a reference lectin. In some If the amount of one or more protein in the test sample is examples the test lectin may include SNA or ConA, while the changed relative to the reference, the subject may be deter reference lectin may include MAL or ECL. In particular mined not to have pre-diabetes or diabetes, respectively. examples, an increase in the ratio of SNA binding to MAL Hence, the amount of one or more protein may provide an binding by a sample (such as an increase of about 2-fold to additional diagnostic criterion for these disorders. about 20-fold) compared to a normal reference standard, such I0121. In some examples, the method may include detect as a reference sample or quantity, may indicate that the Sub ing an increase, such as at least a 2-fold to 10-fold increase ject has pre-diabetes or diabetes. In additional examples, an (such as at least about a 2, 3, 4, 5, 6, 8, or 10-fold increase) in increase in the ratio of SNA binding to MAL binding of about the amount of one or more proteins present in a biological 2-fold to 5-fold (for example, about 2-fold, 3-fold, 4-fold, or sample. In particular examples, the method may include 5-fold) compared to a normal reference standard may indicate detecting an increase in the amount of A1AG and/or A1AT that the Subject has pre-diabetes. In other examples, an protein in a sample from a Subject with pre-diabetes or dia increase in the ratio of SNA binding to MAL binding of about betes, as compared to a control sample. In additional 10-fold to 15-fold (for example, about 10-fold, 11-fold, examples, the method may include detecting an increase or 12-fold, 13-fold, 14-fold, or 15-fold) compared to a normal decrease, Such as at least a 2, 3, 4, or 5-fold increase or reference standard may indicate that the Subject has diabetes. decrease, in the ratio of the amount of A1AG to A1AT in the 0117. In further examples, an increase in the ratio of ConA sample. In particular examples, the ratio of A1AG to A1AT in binding to MAL binding by a sample (such as an increase of a sample from a subject with diabetes may be decreased (Such about 2-fold to about 20-fold) compared to a normal refer as about a 2-fold to 5-fold decrease, for example about a ence standard, Such as a reference sample or quantity, may 2-fold to 3-fold decrease) compared to a control sample. indicate that the subject has pre-diabetes or diabetes. In some I0122. In yet another embodiment, the methods disclosed examples, an increase in the ratio of ConA binding to MAL herein also may include determining the glycosylation pat binding of about 3-fold to 8-fold (for example, about 3-fold, tern of one or more particular proteins (such as proteins 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold) compared to a normal associated with pre-diabetes or diabetes, for example A1AG reference standard may indicate that the Subject has pre or A1AT) in a biological sample. The glycosylation pattern of diabetes. In other examples, an increase in the ratio of ConA a protein may be determined by detecting binding of the binding to MAL binding of about 12-fold to 20-fold (for protein to one or more lectins. In some embodiments, the example, about 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, glycosylation pattern of a protein may be determined by 17-fold, 18-fold, 19-fold, or 20-fold) compared to a normal measuring the binding of the protein in a biological sample to reference standard may indicate that the Subject has diabetes. one or more lectins, such as lectins that detect a type of 0118. Of course, it is not necessary to compare biomol glycosylation of the protein that has been found to be associ ecule glycosylation of a sample directly to glycosylation of ated with pre-diabetes or diabetes. The presence (or change, another sample. Reference standards (such as known quanti Such as increase or decrease) of one or more particular types tative amounts of glycosylation) may be used in lieu of direct of glycosylation as detected by binding to a lectin specific for comparison to results from a reference sample. Hence, in type of glycosylation on the protein may be the glycosylation embodiments, the comparison may be made to a reference pattern of the protein in this example. (which is understood to be results from an actual sample or I0123. In some embodiments, the method may include population of samples, or a qualitative or quantitative refer comparing a glycosylation pattern of one or more proteins in ence that represents a cut-off value, Such as a diagnostic or a test sample (such as urine or saliva) from a subject of interest prognostic level of glycosylation). with a glycosylation pattern of said proteins from a reference standard, such as a reference sample or quantity. In various B. Amount and Glycosylation of Specific Proteins embodiments, if the reference is a normal reference and the 0119. In some embodiments, the methods disclosed herein glycosylation pattern of one or more protein of the test sample also may include determining an amount of one or more is Substantially the same as the glycosylation pattern of said proteins present in a biological sample (such as urine or protein of the reference, the subject may be determined not to saliva). Such as proteins associated with pre-diabetes or dia have pre-diabetes or diabetes. However, if the glycosylation betes. In various embodiments, the methods may include pattern of one or more protein of the test sample is changed comparing an amount of one or more proteins in a test sample relative to the glycosylation pattern of said protein of the (such as urine or saliva) from a Subject of interest with an reference, the subject may be determined to have pre-diabetes amount of said protein in a reference standard, Such as a or diabetes. reference sample or quantity. In embodiments, if the refer 0.124. In another embodiment, if the reference is a pre ence is a normal reference, and the amount of one or more diabetes or diabetes reference, and the glycosylation pattern protein in the test sample is Substantially the same as the of one or more proteins in the test sample is Substantially the amount of said protein in the reference, the Subject may be same as the glycosylation pattern of said proteins in the ref US 2012/0208218 A1 Aug. 16, 2012

erence, then the subject may be determined to have pre I0129 Microvascular diabetic complications include (but diabetes or diabetes, respectively. In embodiments, if the are not limited to) diabetic nephropathy (such as damage to glycosylation pattern of one or more protein in the test sample the kidney which may lead to chronic renal failure), diabetic is changed relative to the reference, the subject may be deter retinopathy (such as growth of friable, poor-quality blood mined not to have pre-diabetes or diabetes, respectively. vessels in the retina which may lead to vision loss), diabetic 0.125. In some examples, the method may include detect neuropathy (such as abnormal and decreased sensation, usu ing an increase, such as at least a 2, 3, 4, or 5 fold increase, in ally in a 'glove and stocking distribution), and diabetic the amount or type of glycosylation of one or more proteins cardiomyopathy (damage to the heart leading to diastolic present in the sample. In a particular example, the method dysfunction and eventually heart failure). may include detecting an increase (such as at least about a 2. 3, 4, or 5-fold increase, for example about a 2-fold or 3-fold 0.130 Macrovascular complications may lead to cardio increase) in the amount of glycosylated A1AG recognized by vascular disease, to which accelerated atherosclerosis may be the lectin ConA in the sample in a subject with pre-diabetes. a contributor. Macrovascular complications include (but are In another particular example, the method may include not limited to) coronary artery disease (such as angina or detecting an increase in the amount of glycosylated Al AG myocardial infarction), stroke, peripheral vascular disease, recognized by the lectin SNA (such as at least about a 2, 3, 4, and diabetic myonecrosis (which may lead to muscle wast or 5-fold increase, for example about a 2-fold or 3-fold ing). increase) in the sample in a Subject with pre-diabetes. In an I0131 Additional diabetic complications may include additional example, the method may include detecting an "diabetic foot' which may lead to skin ulcers, infection, increase in the amount of glycosylated A1AG recognized by necrosis,and/or gangrene. This complication may be due to a the lectin AAL (such as at least about a 2, 3, 4, or 5-fold combination of diabetic neuropathy (such as numbness or increase, for example about a 2-fold or 3-fold increase) in the insensitivity) and vascular damage. Diabetic encephalopathy sample in a subject with diabetes. Thus, in some examples, (such as cognitive decline or dementia) is another diabetic the glycosylation pattern of A1AG may include the amount of complication. A1AG binding to particular lectins (such as ConA, SNA, AAL, or a combination of two or more thereof). (0132. The methods described herein include methods to 0126. Of course, it may not be not necessary to compare determine whether a subject has one or more diabetic com the amount of one or more proteins associated with pre plications. In some embodiments, the method may include diabetes or diabetes of a sample directly to the amount of the detecting an increase (such as at least about a 5-fold increase proteins of another sample. Likewise, it may not be necessary to about 100-fold increase, for example, about a 5-fold, to compare the glycosylation pattern of a protein associated 10-fold, 25-fold, 50-fold, 60-fold, or 100-fold increase) in the with pre-diabetes or diabetes directly to the glycosylation glycosylation profile. Such as the amount of one or more types pattern of the protein of another sample. Reference standards of glycosylation (for example, one or more carbohydrate (such as known quantitative amounts of glycosylation or a groups, such as N-acetylglucosamine, N-acetylgalac particular protein) may be used in various embodiments in tosamine, N-acetylactosamine, N-acetylneuraminic acid, lieu of direct comparison to results from a reference sample. mannose, fucose, or galactose) or an amount of one or more Hence, the comparison may be made to a normal reference oligosaccharide structures (such as a bi-antennary, tri-anten (which may be understood to be results from an actual sample nary, or tetra-antennary structure) present in a sample. In or population of Samples, or a qualitative or quantitative ref Some embodiments, the method may include comparing a erence that represents a cut-off value, such as a diagnostic or glycosylation profile of a test sample (such as urine or saliva) prognostic level of glycosylation or a particular protein). from a subject of interest with a glycosylation profile of a 0127. In additional embodiments, the glycosylation pro reference standard, Such as a reference sample or quantity. In file of a sample from a subject may be determined, and the Some examples, the method may include detecting an amount and/or glycosylation pattern of one or more particular increase in glycosylation recognized by the lectin AAL (Such proteins may be determined (such as sequentially or simulta as at least about 20-fold to 100-fold increase, for example, neously). In one example, the glycosylation profile and the about a 25-fold, 50-fold, 60-fold, or 100-fold increase), such amount of one or more proteins may be determined in a as mannose, in a subject with a diabetic complication. In sample from a subject. In another example, the glycosylation additional examples, the method may include detecting an profile and the glycosylation pattern of one or more proteins increase in glycosylation recognized by the lectin SNA (Such may be determined in a sample from a Subject. In a further as at least about a 20-fold to 100-fold increase, for example, example, the glycosylation profile, and the amount and gly about a 25-fold, 50-fold, 60-fold, or 100-fold increase), such cosylation pattern of one or more proteins may be determined as N-acetylneuraminic acid, in a subject with a diabetic com in a sample from a subject. plication. IV. Methods for Identifying a Subject with Diabetic Compli 0133. In one embodiment, if the reference is a normal cations reference, and the glycosylation profile of the test sample is 0128. In another embodiment, the method may be a Substantially the same as the glycosylation profile of the method to determine ifa Subject has a diabetic complication. normal sample or reference (for example, the amount or type In particular examples, the diabetic complication may be a of glycosylation is Substantially the same), the Subject may be chronic complication. Chronic elevation of blood glucose determined not to have a diabetic complication. However, if level leads to damage of blood vessels (angiopathy) and the glycosylation profile of the test sample is changed relative chronic diabetic complications. The resulting chronic com to the glycosylation profile of the normal sample or reference plications are grouped as “microvascular complications' (for example, the amount of one or more type of glycosylation (due to damage to Small blood vessels) and "macrovascular is increased), the subject may be determined to have a diabetic complications” (due to damage to the arteries). complication. In a particular example, the diabetic complica US 2012/0208218 A1 Aug. 16, 2012 tion may be a microvascular complication, for example, dia example, the glycosylation profile, and the amount and gly betic nephropathy, diabetic retinopathy, or diabetic neuropa cosylation pattern of one or more proteins may be determined thy. in a sample from a Subject. 0134. In another embodiment, if the reference is a diabetic complication reference, and the glycosylation profile of the V. Monitoring test sample is Substantially the same as the reference (for 0.139. In another embodiment, the method may be a example the amount or type of glycosylation is essentially the method to determine whether a therapy is effective for the same. Such as not significantly different), then the Subject treatment of the subject. Thus, in various embodiments, the may be determined to have a diabetic complication. If the method may be performed multiple times over a specified glycosylation profile of the test sample is changed (for time period, Such as days, weeks, months or years. The diag example, a decrease in the amount or type of glycosylation) nostic methods described herein are valuable tools for prac relative to the reference, then the subject may be determined ticing physicians to make quick treatment decisions for dia not to have a diabetic complication. betic conditions, including pre-diabetes and diabetes. These treatment decisions may include the decision to administra 0135) In additional examples, the method for identifying a tion of an anti-diabetic agent, and/or the decision to monitor Subject as having a diabetic complication may include deter a subject for the onset and/or advancement of diabetes. The mining the amount of one or more proteins (such as proteins treatment decisions may also include lifestyle monitoring. associated with diabetic complication, Such as A1AG or The method disclosed herein may also be used to monitor the A1AT) in a sample from the subject. In embodiments, the effectiveness of a therapy. methods may include comparing an amount of one or more 0140. In various embodiments, the glycosylation profile, proteins in a test sample (such as urine or saliva) from a protein expression level, or glycosylation pattern of a sample Subject of interest with an amount of said protein from a from a Subject may be assessed as described above and com reference standard, Such as a reference sample or quantity. In pared to a reference. If the reference, such as a reference particular examples, the method may include detecting an sample or quantity, is a normal reference, and the glycosyla increase in protein amount (Such as at least about a 20-fold tion profile (or amount or glycosylation pattern of a particular increase to about 1000-fold increase, such as about a 20-fold, protein) of the test sample is substantially the same as the 50-fold, 70-fold, 100-fold, 300-fold, 500-fold, or 1000-fold glycosylation profile (or amount or glycosylation pattern of a increase) in a subject with a diabetic complication compared particular protein) of the normal reference, the subject may be to a reference standard, such as a reference sample or quan determined to have an effective therapy Conversely, if the tity. In additional examples, the method may include detect glycosylation profile (or amount or glycosylation pattern of a ing an increase or decrease, such as at least a 2, 3, 4, or 5-fold particular protein) of the test sample is different from the increase or decrease, in the ratio of the amount of A1AG to glycosylation profile (or amount or glycosylation pattern of a A1AT in the sample. In particular examples, the ratio of particular protein) of the normal reference, the subject may be determined to have an ineffective therapy. In embodiments, if A1AG to A1AT in a sample from a subject with a diabetic the reference standard. Such as a reference sample or quantity, complication may be decreased (such as about a 2-fold or is a pre-diabetes or diabetes reference, and the glycosylation 3-fold decrease) compared to a reference standard, such as a profile (or amount or glycosylation pattern of a particular reference sample or quantity. protein) does not differ from the reference, then the subject 0136. In various embodiments, if the reference is a normal may be determined to have an ineffective therapy. Con reference, and the amount of one or more proteins of the test versely, if the glycosylation profile (or amount or glycosyla sample is Substantially the same as the amount of said protein tion pattern of a particular protein) of the test sample is in the normal sample, the Subject may be determined not to different from the reference, the subject may be determined to have a diabetic complication. However, if the amount of one have an effective therapy. or more proteins of the test sample is changed relative to the 0141 Following the assessment of the glycosylation pro amount of said protein of the normal sample or standard, the file, protein expression level, or glycosylation pattern identi Subject is determined to have a diabetic complication. fied herein, the assay results, findings, diagnoses, predictions and/or treatment recommendations may be recorded and/or 0137 In another embodiment, if the reference is a diabetic communicated to technicians, physicians and/or patients, for complication reference, and the amount of one or more pro example. In certain embodiments, computers may be used to tein in the test sample is substantially the same as the amount communicate such information to interested parties. Such as of said protein in the reference, then the subject may be patients and/or the attending physicians. In various embodi determined to have a diabetic complication. If the amount of ments, based on the measurement, the therapy administered one or more proteins in the test sample is changed relative to to a subject may be modified. the reference, the subject is determined not to have a diabetic 0142. In one embodiment, a diagnosis, prediction and/or complication. treatment recommendation based on the glycosylation pro 0.138. In additional embodiments, the glycosylation pro file, protein expression level, or glycosylation pattern in a test file of a sample from a subject may be determined, and the Subject of one or more of the glycosylated biomolecules amount and/or glycosylation pattern of one or more particular described herein may be communicated to the Subject as soon proteins may be determined (such as sequentially or simulta as possible after the assay is completed and the diagnosis neously). In one example, the glycosylation profile and the and/or prediction is generated. In some embodiments, the amount of one or more proteins may be determined in a results and/or related information may be communicated to sample from a subject. In another example, the glycosylation the Subject by the Subject's treating physician. Alternatively, profile and the glycosylation pattern of one or more proteins the results may be communicated directly to a test subject by may be determined in a sample from a Subject. In a further any means of communication, including writing, such as by US 2012/0208218 A1 Aug. 16, 2012

providing a written report, electronic forms of communica tidase-4 inhibitor, but in various other examples, the anti tion, such as email, or telephone. In embodiments, commu diabetic agent may be any agent of interest. nication may be facilitated by use of a computer, Such as in 0145. In embodiments, a therapeutically effective amount case of email communications. In certain embodiments, the ofan anti-diabetic agent may be administered in a single dose, communication containing results of a diagnostic test and/or or in several doses, for example daily, during a course of conclusions drawn from and/or treatment recommendations treatment. The course of treatment may last for any length of based on the test, may be generated and delivered automati time. Such as a day or several days, a week or several weeks, cally to the Subject using a combination of computer hard a month or several months, or a year or several years, so long ware and software which will be familiar to artisans skilled in as the therapeutic effect is observed, such as inhibiting the telecommunications. One example of a healthcare-oriented onset of type II diabetes in a Subject diagnosed with pre communications system is described in U.S. Pat. No. 6,283, diabetes, or inducing a subject diagnosed with type 2 diabetes 761; however, the present methods are not limited to methods or pre-diabetes to a normal glucose tolerance. In various which utilize this particular communications system. In cer embodiments, the Subject may be monitored while undergo tain embodiments of the methods, all or some of the method ing treatment using the methods described herein in order to steps, including the assaying of samples, diagnosing of dis assess the efficacy of the treatment protocol. In this manner, eases, and communicating of assay results or diagnoses, may the length of time or the amount give to the Subject may be be carried out in diverse (e.g., foreign) jurisdictions. modified based on the results obtained using the methods 0143. In several embodiments, identification of a subject disclosed herein. as being pre-diabetic or diabetic may result in the physician 0146 In various embodiments, the therapeutically effec treating the Subject, such as by prescribing an anti-hypergly tive amount of the anti-diabetic agent may depend on the cemic or an anti-diabetic agent to inhibit or delay the onset or anti-diabetic agent being used, the characteristics of the Sub progression of type II diabetes. In additional embodiments, ject being treated (such as age, BMI, physiological condition, the dose or dosing regimen may be modified based on the etc.), the severity and type of the affliction, and the manner of information obtained using the methods disclosed herein. In administration of the agent. The therapeutically effective Some embodiments, the anti-diabetic agent may contain a dose may be determined by various methods, including gen biguanide of the formula: erating an empirical dose-response curve, predicting potency and efficacy by using quantitative structure activity relation ships (QSAR) methods or molecular modeling, and other Formula 1 R methods used in the pharmaceutical Sciences. In certain, non w limiting examples, the therapeutically effective amount of metformin (or a related biguanide analog or homolog) may be R2 --Ni--Ni.N N 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 R3 R4 other, non-limiting examples, the total amount of metformin may be 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 wherein R and R are independently selected from alkyl, three times a day (t.i.d.). In alternative, non-limiting lower alkyl, alkenyl, lower alkenyl, cycloalkyl, aryl, or an examples, the total amount of metformin may be about 500 arylalkyl of the formula: mg or less per day. In embodiments, the Subject may be monitored at different doses of an agent using the assays described herein in order to determineatherapeutically effec Formula 2 tive amount for the subject of interest. 0147 For administration to animals or humans, purified therapeutically active agents are generally combined with a pharmaceutically acceptable carrier. Pharmaceutical prepa rations may contain only one type of anti-diabetic agent, or wherein X is hydrogen or halogen and n=0, 1 or 2; R and R. may be composed of a combination of several types of anti are independently selected from hydrogen, alkyl, lower alkyl, diabetic agents, such as a combination of two or more anti alkenyl, lower alkenyl, cycloalkyl, alkoxy, lower alkoxy, diabetic agents. alkoxyalkyl, and pharmaceutically acceptable salts thereof. 0.148. In general, the nature of the carrier will depend on In particular embodiments, the biguanide antidiabetic agent the particular mode of administration being employed. For may be metformin. Metformin may be manufactured by instance, parenteral formulations usually comprise injectable Lyonnaise Industrielle Pharmaceutique SA (Lyons, France), fluids that include pharmaceutically and physiologically and is also known by its acronym LIPHA SA, and may be acceptable fluids Such as water, physiological saline, bal commercially distributed in the United States as a hydrochlo anced salt Solutions, aqueous dextrose, glycerol or the like as ride salt by the Bristol-Myers Squibb Company (Princeton, a vehicle. For Solid compositions (e.g., powder, pill, tablet, or N.J.) as GLUCOPHAGE(R) XR. Additionally, Bristol-Myers capsule forms), conventional non-toxic Solid carriers may Squibb currently distributes a pharmaceutical having a com include, for example, pharmaceutical grades of mannitol, bination of metformin and glyburide as GLUCOVANCE(R). lactose, starch, or magnesium Stearate. In addition to biologi 0144 Anti-diabetic agents other than biguanides may also cally-neutral carriers, pharmaceutical compositions to be be administered to the identified subject. For example, in administered may contain minor amounts of non-toxic aux alternative embodiments, the anti-diabetic agent may be a iliary Substances, such as wetting or emulsifying agents, pre thiazolidinedione, Such as troglitaZone. In some examples, servatives, and pH buffering agents and the like, for example the anti-diabetic agent may be an incretin or dipeptidyl pep Sodium acetate or Sorbitan monolaurate. US 2012/0208218 A1 Aug. 16, 2012

0149 Anti-diabetic agents may be administered by any simplicifolia lectin II (GSL-2), Hippeastrum hybrid lectin means that achieve their intended purpose. For example, the (HHL), Lycopersicon esculentum lectin (LEL), Lotus tet anti-diabetic agents may be administered to a subject through ragonolobus lectin (LTL), Maackia amurensis lectin I systemic administration, Such as Subcutaneous, intravenous (MAL), Phaseolus vulgaris Agglutinin (PHA-E), Sambucus or intraperitoneal administration, by Suppository, or by oral nigra lectin (SNA), and Vicia villosa lectin (VVL). The bind administration. In embodiments, the anti-diabetic agent may ing specificity of exemplary lectins is presented in Table 1, be administered alone or in combination with another anti however, the table is not exhaustive and the lectins may also diabetic agent. In certain embodiments, the anti-diabetic bind to additional carbohydrate modifications. agent may be administered in the absence of administering any other anti-diabetic agent. TABLE 1 0150. In embodiments, other measures may be taken to inhibit or delay the onset of type II diabetes in subjects at a Exemplary lectins and binding specificity heightened risk of developing the disease. For example, in Lectin Binding specificity Some embodiments, a subject may be instructed, trained, or induced to adopt anti-diabetic lifestyle modifications. For Aletiria aurantialectin (AAL) Fucose Concanavalin A (ConA) Mannose-glucose example, the Subject may be counselled to reduce caloric Datura stramonium lectin (DSL) Gal B14 GlcNAc, oligomers intake or to exercise. In various embodiments, the methods of GlcNAc and LacNAc disclosed herein may be used to monitor the effectiveness of Erythrina cristagali lectin (ECL) B-gal (1->4)-GlcNAc; galactose and galactosides these alternative measures, for instance, to determine if phar Griffonia simplicifolia lectin II (GSL-2) terminal C-D-galactosyl maceutical intervention is warranted for a Subject of interest. residues, terminal N-acetyl-C-D- galactosaminyl residues VI. Lectins Hippeastrum hybrid lectin (HHL) Mannose Lycopersicon escientain lectin (LEL) GlcNAc oligomers 0151. Lectins are carbohydrate-binding proteins, some of Lotus tetragonolobits lectin (LTL) Fucose which are specific for particular carbohydrate moieties. The Maackia amurensis lectin I (MAL) N-acetylneuraminic acid (sialic acid) term “lectin’ was originally coined to define agglutinins Phaseolus vulgaris Agglutinin (PHA-E) Gal-B1.4 GlcNAc which could discriminate among types of red blood cells, Sambucus nigra lectin (SNA) C-NeuNAc-2->6)-Gal, however the term is now used more generally and includes C-NeuNAc-2->6)-GalNAc, carbohydrate-binding proteins from many sources regardless C-NeuNAc-2->3)-Gal of their ability to agglutinate cells. Lectins have been found in Vicia villosa lectin (VVL) O-linked GalNAc plants, viruses, microorganisms and animals. Their function in nature is unclear, however lectins share the common prop 0.155. Additional lectins are well known to one of skill in erty of binding to defined Sugar structures. the art. In some examples, lectins may include Agaricus 0152. In embodiments, the glycosylation profile of a bisporus lectin, Amaranthus caudatus lectin, Grifonia sim sample may be determined by binding to one or more lectins. plicifolia lectin I, Bauhinia purpurea alba lectin, Codium In some embodiments, the glycosylation profile may be fragile lectin, Dolichos biflorus lectin, Erythrina corallden expressed in terms of the lectins to which a sample binds dron lectin, Euonymos europaeus lectin, Glycine max lectin, (such as the lectins provided in Table 1, below). The glyco Helix aspersa lectin, Helix pomatia lectin, Maclura ponifera Sylation profile may also be expressed in terms of the particu lectin, Narcissus pseudonarcissus lectin, Phaseolus coc lar carbohydrate modifications present in the sample, based cineus lectin, Phaseolus vulgaris Llectin, Phytolacca Ameri on the carbohydrate-binding specificity of the particular lec cana lectin, Pisum sativum lectin, Psophocarpus tet tins (such as described in Table 1, below) to which the sample ragonolobus I lectin, Solanum tuberosum lectin, Sophora binds. However, in embodiments, the identification of spe japonica terminal lectin, Wisteria floribunda lectin, Anguilla cific carbohydrates in a sample may not be essential to deter anguilla lectin, Arachis hypogaea lectin, Artocarpus integri mining the glycosylation profile of the sample in the methods folia lectin, Bandeiraea simplicifolia lectin, Caragana described herein. arborescens lectin, Cicer arietinum lectin, Galanthus nicalis 0153. Similarly, in some embodiments, the glycosylation lectin, Lens culinaris lectin, Limulus polyphemus lectin, pattern of a particular protein may be determined by binding Pseudomonas aeruginosa lectin, Ricin communis agglutinin, to one or more lectins. In some embodiments, the glycosyla Triticum vulgaris lectin, Ulex europaeus lectin, Vicia faba tion pattern of a protein may be expressed in terms of the lectin, and Visum album lectin. In various embodiments, any lectins to which the protein binds (such as the lectins provided lectin that identifies a difference between the glycosylation in Table 1, below). The glycosylation pattern of the protein profile of a sample or the glycosylation pattern of one or more may also be expressed in terms of the particular carbohydrate protein from a Subject with pre-diabetes, diabetes, or a dia modifications present on the protein, based on the carbohy betic complication and a reference may be used in the dis drate-binding specificity of the particular lectins (such as closed methods. described in Table 1, below) to which the protein binds, or by other methods known in the art (Such as mass spectrometry). VII. Detection Methods However, in embodiments, the identification of specific car bohydrates on the protein may not be essential to determining 0156 The methods disclosed herein may be performed in the glycosylation pattern of the protein in the methods the form of various assay formats, which are well known in described herein. the art. In some examples, the assays may be antibody-based 0154 Lectins are well known to those of skill in the art. In or lectin-based assays. There are two main types of assays, particular examples, lectins may include Aleuria aurantia homogenous and heterogenous. In homogenous assays, the lectin (AAL), Concanavalin A (ConA), Datura stramonium immunological reaction between an antigen and an antibody lectin (DSL), Erythrina cristagalli lectin (ECL), Grifonia (or the reaction between an oligosaccharide and a lectin) and US 2012/0208218 A1 Aug. 16, 2012

the detection are carried out in a homogenous reaction. Het the bound antigen. Any excess unbound enzyme-linked lectin erogenous assays include at least one separation step, which is washed away after the reaction. The substrate for the allows the differentiation of reaction products from unreacted enzyme used in the assay is then added and the reaction reagents. between the substrate and the enzyme produces a color 0157 ELISA is a heterogenous assay, which has been change. The amount of visual color change is a direct mea widely used in laboratory practice since the early 1970s, and Surement of specific enzyme-conjugated bound lectin, and may be used in the methods disclosed herein. The assay may consequently the carbohydrate modification present on the be used to detect protein antigens or carbohydrates (such as particular antigen (the glycosylation pattern of the antigen) in monosaccharides or oligosaccharides linked to a protein) in the sample tested. various formats. In the “sandwich' format the antigen or 0.161 ELISA may also be used as a competitive assay. In carbohydrate being assayed is held between two different the competitive assay format, the test specimen containing antibodies or lectins, respectively. Another type of sandwich the antigen or oligosaccharide to be determined is mixed with assay holds the glycoprotein between an antibody and a lec a precise amount of enzyme-labeled antigen or oligosaccha tin. ride (such as an enzyme-labeled glycoprotein containing the 0158. A sandwich ELISA may be used to detect the pres oligosaccharide) and both compete for binding to an anti ence or amount of an antigen in a sample, such as a glyco antigen antibody or a lectin attached to a solid surface. Excess protein (a “protein ELISA). In this method, a solid surface is free enzyme-labeled antigen or glycoprotein is washed off first coated with a solid phase antibody. The test sample, before the substrate for the enzyme is added. The amount of containing the antigen (such as a diagnostic glycoprotein), or color intensity resulting from the enzyme-Substrate interac a composition containing the antigen, Such as a urine or saliva tion is a measure of the amount of antigen or oligosaccharide sample from a Subject of interest, is then added and the in the sample tested. antigen is allowed to react with the bound antibody. Any 0162 Similar assays may be performed using alternate unbound antigen is washed away. A known amount of platforms for the identification of the glycosylation profile, enzyme-labeled antibody is then allowed to react with the amount of particular proteins, or glycosylation pattern of bound antigen. Any excess unbound enzyme-linked antibody particular proteins. For example, microsphere-based assays is washed away after the reaction. The substrate for the may be used to detect binding of an antigen to an antibody or enzyme used in the assay is then added and the reaction a carbohydrate modification to a lectin. Briefly, microsphere between the substrate and the enzyme produces a color beads are coated with an antibody or a lectin and mixed with change. The amount of visual color change is a direct mea a sample, such that an antigen or a carbohydrate modification, Surement of specific enzyme-conjugated bound antibody, and respectively, present in the sample that are specifically reac consequently the antigen present in the sample tested. tive with the antibody or lectin bind to the bead. The bead 0159 ELISA may also be used to detect the presence or bound complexes are allowed to react with fluorescent-dye amount carbohydrates (such as monosaccharides or oligosac labeled antibody or lectin, and are measured using a micro charides, for example in the form of glycoproteins or gly sphere reader (such as a LumineX instrument). Alternatively, colipids) in a sample by utilizing a lectin in place of an the antibody or lectin may be coupled to a magnetic bead, antibody in the assay. In a “lectin ELISA.” a solid surface is allowing separation and detection of particular proteins or coated with a lectin. The test sample containing one or more particular carbohydrate modifications present in the sample. glycosylated biomolecule, such as a urine or saliva sample 0163. In additional examples, the glycosylation profile, from a subject of interest, is then added and the carbohydrate amount of particular proteins, or glycosylation pattern of in the glycoprotein is allowed to react with the bound lectin. particular proteins may be determined with methods utilizing Any unbound glycosylated biomolecule is washed away and antibody or lectin arrays. For example, the glycosylation pro a known amount of enzyme-labeled lectin is then allowed to file of a sample may be determined by applying the sample to react with the bound antigen. Any excess unbound enzyme a lectin array including two or more lectins (for example, two linked lectin is washed away after the reaction. The substrate or more of PHA-E, LEL, AAL, ConA, SNA, or MAL) at for the enzyme used in the assay is then added and the reaction addressable locations and detecting binding of the sample to between the substrate and the enzyme produces a color particular lectins. The profile of binding of a sample from a change. The amount of visual color change is a direct mea Subject to the array may be compared to a reference profile Surement of specific enzyme-conjugated bound lectin, and (such as a profile from a normal subject) to determine whether consequently the carbohydrate present in the sample tested. A the Subject is pre-diabetic, diabetic, or has a diabetic compli lectin ELISA may not identify the particular biomolecule(s) cation. Similarly, the amount of particular proteins present in which is recognized by the lectin, rather a lectin ELISA may a sample may be determined by applying the sample to an provide a glycosylation profile, such as the amount or type of antibody array including two or more antibodies (for example one or more carbohydrate modifications on one or more bio A1AG and A1AT) and detecting binding of the sample to molecule that is present in the sample. particular antibodies. In additional examples, the glycosyla 0160. In other examples, an “antibody-lectin ELISA may tion pattern of particular proteins (for example, Al AG or be used to detect the presence or amount of carbohydrate A1AT) may be determined by applying a sample containing modifications on a particular protein in a sample. In this the protein to a lectin array (Such as Qproteome Glyco Array, method, a solid surface is first coated with a solid phase Qiagen, Valencia, Calif.) and detecting binding to particular antibody. The test sample, containing the antigen (Such as lectins in the array with a protein-specific antibody. Al AG or A1AT), or a composition containing the antigen, 0164. Other methods for determining the glycosylation Such as a urine or saliva sample from a Subject of interest, is profile, amount of glucosylated proteins, or glycosylation then added and the antigen is allowed to react with the bound pattern of particular proteins may be used. For example, antibody. Any unbound antigen is washed away. A known Western blotting may be used to determine the amount of a amount of enzyme-labeled lectin is then allowed to react with particular protein in a sample. Similarly, lectin blotting may US 2012/0208218 A1 Aug. 16, 2012

be used to determine the amount of a particular carbohydrate vides diagnostic information to the user. The binding of one modification present in a sample, thus providing a glycosy or more proteins in the sample to the lectins present in the test lation profile of the sample. The glycosylation pattern of a device may be detected, and the glycosylation profile of the protein may be determined, for example by immunoprecipi test Subject may be compared to the glycosylation profile of a tation of a particular protein from a sample, followed by lectin control sample, wherein a change in the glycosylation profile blotting to determine the carbohydrate modifications present of the sample from the test subject as compared to the control on the protein. sample indicates that the Subject has pre-diabetes, diabetes, or a diabetic complication (Such as diabetic nephropathy). VIII. Capture Device Methods 0169. In some examples, the methods disclosed herein 0165. In various embodiments, the disclosed methods may further include determining the amount of one or more may be carried out using a sample capture device. Such as a proteins (such as one or more protein associated with pre lateral flow device (for example a lateral flow test strip) that diabetes or diabetes) in a biological sample (such as saliva or allows detection of a glycosylation profile, glycosylation pat urine) from a test Subject by applying the biological sample to tern, or amount of protein. a lateral flow test device. The test device may include one or 0166 Point-of-use analytical tests have been developed more antibodies (such as anti-A1 AG or anti-A1AT) at an for the routine identification or monitoring of health-related addressable location. In embodiments, the binding of the pro conditions (such as pregnancy, cancer, endocrine disorders, teins in the sample from the test subject to the particular infectious diseases or drug abuse) using a variety of biologi antibodies may be detected, and the amount of the protein in cal samples (such as urine, serum, plasma, blood, saliva). the sample from the test subject may be compared to the Some of the point-of-use assays are based on highly specific amount of the protein in a control sample, wherein a change interactions between specific binding pairs, such as antigen/ in the amount of the protein in the sample from the test subject antibody, hapten/antibody, lectin/carbohydrate, apoprotein/ as compared to the control sample may indicate that the cofactor and biotin/(strept)avidin. The assays are often per Subject has pre-diabetes, diabetes, or a diabetic complication formed with test strips in which a specific binding pair (such as diabetic nephropathy). member is attached to a mobilizable material (such as a metal A. Flow-Through devices Sol or beads made of latex or glass) or an immobile Substrate 0170 Flow-through type assay devices were designed, in (such as glass fibers, cellulose strips or nitrocellulose mem part, to obviate the need for incubation and washing steps branes). Particular examples of some of these assays are associated with dipstick assays. In embodiments, flow shown in U.S. Pat. Nos. 4,703,017; 4,743,560; and 5,073,484. through immunoassay devices may include a capture reagent The test strips may include a flow path from an upstream (such as one or more lectins) bound to a porous membrane or sample application area to a test site. For example, the flow filter to which a liquid sample is added. As the liquid flows path may be from a sample application area through a mobi through the membrane, target analyte (Such as glycoproteins, lization Zone to a capture Zone. The mobilization Zone may glycolipids, or proteins) binds to the capture reagent. The contain a mobilizable marker that interacts with an analyte or addition of sample is followed by (or made concurrent with) analyte analog, and the capture Zone may contain a reagent addition of detector reagent (such as, labeled (e.g., gold that binds the analyte or analyte analog to detect the presence conjugated, or colored latex particle-conjugated) glycopro of an analyte in the sample. tein, labeled (e.g., gold-conjugated or colored latex particle 0167 Examples of migration assay devices, which usually conjugated) glycolipid or labeled (e.g., gold-conjugate or incorporate within them reagents that have been attached to colored latex particle-conjugated) protein (such as A1AT or colored labels, thereby permitting visible detection of the A1AG). Alternatively, the detector reagent may be placed on assay results without addition of further Substances, are the membrane in a manner that permits the detector to mix found, for example, in U.S. Pat. No. 4,770.853; WO with the sample and thereby label the analyte. The visual 88/08534; and EP-A 0299428. There are a number of com detection of detector reagent provides an indication of the mercially available lateral-flow type tests and patents disclos presence of target analyte in the sample. Representative flow ing methods for the detection of large analytes (MW greater through assay devices are described in U.S. Pat. Nos. 4,246, than 1,000 Daltons) as the analyte flows through multiple 339; 4,277,560; 4,632,901; 4,812,293: 4,920,046; and 5,279, Zones on a test strip. Examples are found in U.S. Pat. No. 935; and U.S. Patent Application Publication Nos. 5.229,073 (measuring plasma lipoprotein levels), and U.S. 2003.0049857 and 20040241876. Migration assay devices Pat. Nos. 5,591,645; 4,168,146; 4,366,241; 4.855,240; 4,861, usually incorporate within them reagents that have been 711; 5,120,643; European Patent No. 0296724; WO attached to colored labels, thereby permitting visible detec 97/06439; and WO 98/36278. Multiple Zone lateral flow test tion of the assay results without addition of further sub strips are disclosed in U.S. Pat. No. 5,451,504, U.S. Pat. No. stances. See, for example, U.S. Pat. No. 4,770.853; PCT 5,451,507, and U.S. Pat. No. 5,798,273. U.S. Pat. No. 6,656, Publication No. WO 88708534 and European Patent No. 744 discloses a lateral flow test strip in which a label binds to EP-AO 299 428. an antibody through a streptavidin-biotin interaction. 0171 There are a number of commercially available lat 0.168. In particular examples, the methods disclosed eral flow type tests and patents disclosing methods for the herein may include application of a biological sample (Such detection of large analytes (MW greater than 1,000 Daltons). as saliva or urine) from a test subject to a lateral flow test U.S. Pat. No. 5.229,073 describes a semiquantitative com device for the detection of a glycosylation profile of the petitive immunoassay lateral flow method for measuring sample. In embodiments, the lateral flow test device may plasma lipoprotein levels. This method utilizes a plurality of include one or more lectins (such as one or more of PHA-E, capture Zones or lines containing immobilized antibodies to LEL, AAL, DSL, ConA, SNA, or MAL) at one or more bind both the labeled and free lipoprotein to give a semi addressable locations. The addressable locations may be, for quantitative result. In addition, U.S. Pat. No. 5,591,645 pro example, a linear array or other geometric pattern that pro vides a chromatographic test strip with at least two portions. US 2012/0208218 A1 Aug. 16, 2012

The first portion includes a movable tracer and the second to assay to optimize the immunoassay results. The fluid portion includes an immobilized binder capable of binding to migrates distally through all the functional regions of the the analyte. Additional examples of lateral flow tests for large strip. The final distribution of the fluid in the individual func analytes are disclosed in the following patent documents: tional regions depends on the adsorptive capacity and the U.S. Pat. Nos. 4,168,146; 4,366,241; 4.855,240; 4,861,711; dimensions of the materials used. and 5,120,643; European Patent No. 0296724; WO 0.174. In some embodiments, porous solid supports, such 97/06439; and WO 98/36278. There are also lateral flow type as nitrocellulose, described hereinabove are preferably in the tests for the detection of small analytes (MW 100-1,000 Dal form of sheets or strips. The thickness of such sheets or strips tons). Generally, these small analyte tests involve “typical may vary within widelimits, for example, from about 0.01 to competitive inhibition to produce negative or indirect report 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 ing results (i.e., reduction of signal with increasing analyte mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, concentration), as exemplified by U.S. Pat. No. 4.703,017. or from about 0.11 to 0.15 mm. The pore size of such sheets However, several approaches have been developed for detect or strips may similarly vary within wide limits, for example ing Small analytes using lateral flow tests that produce posi from about 0.025 to 15 microns, or more specifically from tive or direct reporting results (i.e., increase in signal with about 0.1 to 3 microns; however, pore size is not intended to increasing analyte concentration). These include, for be a limiting factor in selection of the solid support. The flow instance, U.S. Pat. Nos. 5,451,504; 5,451,507; 5,798,273: rate of a Solid Support, where applicable, may also vary within and 6,001,658. U.S. Pat. No. 5,451,504 provides a method widelimits, for example from about 12.5 to 90 sec/cm (i.e., 50 with three specific Zones (mobilization, trap and detection) to 300 sec/4 cm), about 22.5 to 62.5 sec/cm (i.e., 90 to 250 each containing a different latex conjugate to yield a positive sec/4 cm), about 25 to 62.5 sec/cm (i.e., 100 to 250 sec/4 cm), signal. The mobilization Zone contains labeled antibody or about 37.5 to 62.5 sec/cm (i.e., 150 to 250 sec/4 cm), or about lectin to bind the analyte in the sample. In the trap Zone, 50 to 62.5 sec/cm (i.e., 200 to 250 sec/4 cm). In specific unbound, labeled antibody or lectin is then trapped by immo embodiments of devices described herein, the flow rate is bilized analyte analog. The detection Zone captures the about 62.5 sec/cm (i.e., 250 sec/4 cm). In other specific labeled analyte-antibody or lectin complex. U.S. Pat. No. embodiments of devices described herein, the flow rate is 5,451.507 describes a two-Zone, disconnected assay method. about 37.5 sec/cm (i.e., 150 sec/4 cm). The first Zone has non-diffusively bound reagent that binds 0.175. Another common feature to be considered in the use with a component, for example, an analyte analog bound to, of assay devices is a means to detect the formation of a or capable of becoming bound to, a member of a signal complex between an analyte (such as a glycoprotein or a producing system. The second Zone binds to the component glycolipid or one or more particular proteins) and a capture only when the analyte to be tested is present. The distance the reagent (Such as one or more lectins orantibodies). A detector component migrates into the second Zone is directly related to (also referred to as detector reagent) serves this purpose. A the concentration of analyte. U.S. Pat. No. 5,798.273 dis detector may be integrated into an assay device (for example closes a lateral flow device that includes a capture Zone with included in a conjugate pad, as described below), or may be immobilized analyte analog and one or more read-out Zones applied to the device from an external source. to bind labeled analyte-analog. U.S. Pat. No. 6,001,658 dis 0176 A detector may be a single reagent or a series of closes a test strip device with a diffusible, labeled binding reagents that collectively serve the detection purpose. In some partner that binds with analyte, an immobilized analyte, and a instances, a detector reagent is a labeled binding partner detection area containing an immobilized antibody. specific for the analyte (such as, gold-conjugated lectin for a 0172 Devices described herein generally include a strip of glycoprotein analyte or a glycolipid analyte or a gold-conju absorbent material (Such as a microporous membrane), gated antibody for a particular protein). Exemplary lectins are which, in Some instances, may be made of different Sub listed in Table 1 above (for example, PHA-E, LEL, AAL, stances each joined to the other in Zones, which may be DSL, ConA, SNA, and/or MAL). Exemplary antibodies abutted and/or overlapped. In some examples, the absorbent include, but are not limited to antibodies to A1AG and A1AT. strip may be fixed on a Supporting non-interactive material Such antibodies may be commercially available. Exemplary (such as nonwoven polyester), for example, to provide commercially available antibodies include A1AG antibodies increased rigidity to the strip. Zones within each strip may (such as catalog numbers sc-59447, sc-51018, sc-51020, differentially contain the specific binding partner(s) and/or Santa Cruz, Biotechnology, Santa Cruz, Calif.; catalog num other reagents required for the detection and/or quantification ber AO011, Dako, Carpinteria, Calif.; catalog numbers ab440, of the particular analyte being tested for, for example, glyco ab17695, Abcam, Cambridge, Mass.) and A1AT antibodies proteins and/or glycolipids or particular proteins. Thus these (such as catalog numbers sc-59435, sc-59436, sc-69986, Zones may be viewed as functional sectors or functional Santa Cruz, Biotechnology, Santa Cruz, Calif.; catalog num regions within the test device. bers ab7633, ab9400, ab14226, Abcam, Cambridge, Mass.). 0173. In general, a fluid sample is introduced to the strip at 0177. In other instances, a detector reagent collectively the proximal end of the strip, for instance by dipping or includes an unlabeled first binding partner specific for the spotting. A sample is collected or obtained using methods analyte and a labeled second binding partner specific for the well known to those skilled in the art. The sample containing first binding partner and so forth. Thus, the detector may be a the glycoproteins and/or glycolipids or particular proteins to labeled lectin or antibody specific for a glycoprotein or a be detected may be obtained from any biological source. glycolipid or a particular protein. The detector may also be an Examples of biological sources include blood serum, blood unlabeled first lectin orantibody specific for the glycoprotein plasma, urine, spinal fluid, saliva, fermentation fluid, lymph or glycolipid or a particular protein and a labeled second fluid, tissue culture fluid and ascites fluid of a human or antibody that specifically binds the unlabeled first lectin or animal. The sample may be diluted, purified, concentrated, antibody. In each instance, a detector reagent specifically filtered, dissolved, suspended or otherwise manipulated prior detects bound analyte of an analyte-capture reagent complex US 2012/0208218 A1 Aug. 16, 2012

and, therefore, a detector reagent preferably does not Substan 4,945,042; 4,496,654; 5,001,049; 5,075,078; 5,126,241: tially bind to or react with the capture reagent or other com 5,451,504; 5,424, 193; 5,712,172: 6,555,390; 6,258,548; ponents localized in the analyte capture area. Such non-spe 6,699,722; and 6,368,876; EP 0810436; and WO92/12428; cific binding or reaction of a detector may provide a false WO 94/01775; WO95/16207; and WO 97/06439, each of positive result. Optionally, a detector reagent may specifically which is incorporated by reference. recognize a positive control molecule (such as a non-specific 0182 Many lateral flow devices are one-step lateral flow human IgG for a labeled Protein A detector, or a labeled assays in which a biological fluid is placed in a sample area on Protein G detector, or a labeled anti-human Ab(Fc)) that is abibulous strip (though, non-bibulous materials may be used, present in a secondary capture area. and rendered bibulous, e.g., by applying a Surfactant to the material), and allowed to migrate along the strip until the B. Flow-Through Device Construction and Design liquid comes into contact with a specific binding partner 0.178 A flow-through device may involve a capture (such as a lectin or antibody) that interacts with an analyte reagent (such as one or more lectins) immobilized on a Solid (such as one or more glycoprotein, glycolipid, or particular Support, typically, microtiter plate or a membrane (Such as, protein) in the liquid. Once the analyte interacts with the nitrocellulose, nylon, or PVDF). Characteristics of useful binding partner, a signal (such as a fluorescent or otherwise membrane have been previously described; however, it is visible dye) indicates that the interaction has occurred. Mul useful to note that in a flow-through assay, capillary rise is not tiple discrete binding partners (such as lectins or antibodies) a particularly important feature of a membrane as the sample may be placed on the strip (for example in parallel lines) to moves vertically through the membrane rather than across it detect multiple analytes (such as glycoproteins, glycolipids, as in a lateral flow assay. In a simple representative format, the or particular proteins) in the liquid. The test strips may also membrane of a flow-through device is placed in functional or incorporate control indicators, which provide a signal that the physical contact with an absorbent layer (see, e.g., descrip test has adequately been performed, even if a positive signal tion of “absorbent pad' below), which acts as a reservoir to indicating the presence (or absence) of an analyte is not seen draw a fluid sample through the membrane. Optionally, fol on the Strip. lowing immobilization of a capture reagent, any remaining 0183 The construction and design of lateral flow devices protein-binding sites on the membrane may be blocked (ei is very well known in the art, as described, for example, in ther before or concurrent with sample administration) to Millipore Corporation, A Short Guide Developing immuno minimize nonspecific interactions. chromatographic Test Strips, 2nd Edition, pp. 1-40, 1999, 0179. In operation of a flow-through device, a fluid sample available by request at (800) 645-5476; and Schleicher & (such as a bodily fluid sample) is placed in contact with the Schuell, Easy to Work with BioScience, Products and Proto membrane. Typically, a flow-through device also includes a cols 2003, pp. 73-98, 2003, 2003, available by request at sample application area (or reservoir) to receive and tempo Schleicher & Schuell BioScience, Inc., 10 Optical Avenue, rarily retain a fluid sample of a desired volume. The sample Keene, N.H.03431, (603)352-3810; both of which are incor passes through the membrane matrix. In this process, an porated herein by reference. analyte in the sample (such as a glycoprotein or glycolipid or 0.184 Lateral flow devices have a wide variety of physical one or more particular protein) may specifically bind to the formats that are equally well known in the art. Any physical immobilized capture reagent (Such as one or more lectins or format that Supports and/or houses the basic components of a antibodies). Where detection of an analyte-capture reagent lateral flow device in the proper function relationship is con complex is desired, a detector reagent (Such as labeled lectin templated by this disclosure. or labeled antibodies that specifically bind glycoproteins, 0185. The basic components of a particular embodiment glycolipids, or one or more particular protein) may be added of a lateral flow device are illustrated in FIG. 1A, which with the sample or a solution containing a detector reagent shows a particular embodiment of a bibulous lateral flow strip may be added Subsequent to application of the sample. If an 12. Lateral flow strip 12 is divided into a proximal sample analyte is specifically bound by capture reagent, a visual application pad 14, an intermediate test result Zone 16, and a representative attributable to the particular detector reagent distal absorbent pad 18. Flow strip 12 is interrupted by a may be observed on the surface of the membrane. Optional conjugate pad 19 that contains labeled conjugate (such as wash steps may be added at any time in the process, for gold- or latex-conjugated lectin, antibody, or glycoprotein). A instance, following application of the sample, and/or follow flow path along strip 12 passes from proximal pad 14, through ing application of a detector reagent. conjugate pad 19, into test result Zone 16, for eventual col lection in absorbent pad 18. Selective binding agents are C. Lateral Flow Device Construction and Design positioned on a proximal test line 20 in test result membrane 16. A control line 22 is provided intest result Zone 16, slightly 0180 Lateral flow devices are commonly known in the art. distal to test line 20. FIG. 1B illustrates a housing 10 for test Briefly, a lateral flow device is an analytical device having as strip 12 that includes a rubber syringe dam 24 distal to the its essence a test strip, through which flows a test sample fluid proximal sample application pad 14, a Sufficiency indicator that is suspected of containing an analyte of interest. The test guide 26, a Volumetric indicator 28 distal to the guide, a fluid and any Suspended analyte may flow along the strip to a results window 30, and an grip area 32. detection Zone in which the analyte (if present) interacts with 0186. In operation of the particular embodiment of a lat a capture agent and a detection agent to indicate a presence, eral flow device illustrated in FIG. 1A, a fluid sample con absence and/or quantity of the analyte. taining an analyte of interest, such as one or more glycopro 0181 Numerous lateral flow analytical devices have been tein, glycolipid, or particular protein of interest, is applied to disclosed, and include those shown in U.S. Pat. Nos. 4.313, the sample pad 14. In some examples, the sample may be 734: 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4.806,311; applied to the sample pad 14 by dipping the end of the device 4,806,312; 4,861,711; 4,855,240; 4,857,453: 4,943,522; containing the sample pad 14 into the sample (such as urine or US 2012/0208218 A1 Aug. 16, 2012

saliva) or by applying the sample directly onto the sample pad analyte to be detected, the sample volume, the desired flow 14 (for example by placing the sample pad 14 in the mouth of rate and others, and may routinely select the useful materials the subject). In other examples where a sample is whole accordingly. blood, an optional developer fluid is added to the blood sample to cause hemolysis of the red blood cells and, in some TABLE 2 cases, to make an appropriate dilution of the whole blood sample. Exemplary lateral flow device component materials 0187. From the sample pad 14, the sample passes, for Component Useful Material instance by capillary action, to the conjugate pad 19. In the Sample Pad Glass fiber conjugate pad 19, the analyte of interest, Such as a glycopro Woven fibers Screen tein, glycolipid, or particular protein, may bind (or be bound Non-woven fibers by) a mobilized or mobilizable detector reagent, such as a Cellulosic filters glycoprotein (for example fetuin or an affinity-purified lectin Paper Conjugate Pad Glass fiber binding sample fraction), a lectin (for example one or more of Polyester PHA-E, LEL, AAL, DSL, ConA, SNA, or MAL) or an anti Paper body (such as antibody that recognizes A1AG or A1AT). For Surface modified polypropylene example, a glycoprotein analyte may bind to a labeled (e.g., Membrane Nitrocellulose (including pure nitrocellulose and modified gold-conjugated or colored latex particle-conjugated) glyco nitrocellulose) protein, lectin orantibody contained in the conjugatepad. The Nitrocellulose direct cast on polyester analyte complexed with the detector reagent may Subse Support Polyvinylidene fluoride quently flow to the test result Zone 16 where the complex may Nylon further interact with an analyte-specific binding partner (Such Absorbent Pad Cellulosic filters as a lectin, an antibody that binds the lectin, a glycoprotein or Paper other particular protein, an anti-hapten antibody, or strepta vidin), which is immobilized at the proximal test line 20. In Some examples, a lectin complexed with a detector reagent 1. Sample Pad (such as, gold-conjugated lectin or antibody, labeled (e.g., gold-conjugated) antibody may further bind to unlabeled, (0190. The sample pad (such as sample pad 14 in FIG. 1A) is a component of a lateral flow device that initially receives oxidized antibodies or lectins immobilized at the proximal the sample, and may serve to remove particulates from the test line 20. The formation of a complex, which results from sample. Among the various materials that may be used to the accumulation of the label (e.g., gold or colored latex) in construct a sample pad (see Table 2), a cellulose sample pad the localized region of the proximal test line 20 is detected. may be beneficial if a large bed volume (e.g., 250 ul/cm) is a The control line 22 may contain an immobilized, detector factor in a particular application. Sample pads may be treated reagent-specific binding partner, which may bind the detector with one or more release agents, such as buffers, salts, pro reagent in the presence or absence of the analyte. Such bind teins, detergents, and Surfactants. Such release agents may be ing at the control line 22 indicates proper performance of the useful, for example, to promote resolubilization of conjugate test, even in the absence of the analyte of interest. The test pad constituents, and to block non-specific binding sites in results may be visualized directly, or may measured using a other components of a lateral flow device, Such as a nitrocel reader (such as a scanner). The reader device may detect color lulose membrane. Representative release agents include, for or fluorescence from the readout area (for example, the test example, trehalose or glucose (1%-5%), PVP or PVA (0.5%- line and/or control line). 2%), Tween 20 or Triton X-100 (0.1%-1%), casein (1%-2%), 0188 In another embodiment of a lateral flow device, SDS (0.02%-5%), and PEG (0.02%-5%). there may be a second (or third, fourth, or more) test line located parallel or perpendicular (or in any other spatial rela 2. Membrane and Application Solution: tionship) to test line 20 in test result Zone 16 (for exampletest 0191 The types of membranes useful in a lateral flow lines 20a, 20b, and 20c in FIG. 1C). The operation of this device (such as nitrocellulose, nylon and PVDF), and consid particular embodiment is similar to that described in the erations for applying a capture reagent to Such membranes immediately preceding paragraph with the additional consid have been discussed previously. erations that (i) a second detector reagent specific for a second analyte. Such as another lectin or antibody, may also be con 3. Conjugate Pad tained in the conjugate pad, and (ii) the second test line will contain a second specific binding partner having affinity for a 0.192 The conjugate pad (such as conjugatepad 19 in FIG. second analyte. Such as a second glycoprotein or glycolipid or 1A) serves to, among other things, hold a detector reagent. In a particular protein in the sample. Similarly, if a third (or Some embodiments, a detector reagent may be applied exter more) test line is included, the test line will containa third (or nally, for example, from a developer bottle, in which case a more) specific binding partner having affinity for a third (or lateral flow device need not contain a conjugate pad (see, for more) analyte. example, U.S. Pat. No. 4,740,468). 0189 Some of the materials that may be useful for the 0193 Detector reagent(s) contained in a conjugate pad are components of a lateral flow device are shown in Table 2. typically released into Solution upon application of the test However, one of skill in the art will recognize that the par sample. A conjugate pad may be treated with various Sub ticular materials used in a particular lateral flow device will stances to influence release of the detector reagent into solu depend on a number of variables, including, for example, the tion. For example, the conjugate pad may be treated with PVA US 2012/0208218 A1 Aug. 16, 2012 20 or PVP (0.5% to 2%) and/or Triton X-100 (0.5%). Other three months, six months, or more. The glycosylation profile release agents include, without limitation, hydroxypropylm of a sample (such as urine or saliva) from the Subject is ethyl cellulose, SDS, Brij and B-lactose. A mixture of two or determined, for example by determining sample binding to more release agents may be used in any given application. In one or more lectins. A compound may be considered to a particular disclosed embodiment, the detector reagent in decrease or block one or more type of glycosylation associ conjugate pad 19 is gold-conjugated lectin or antibody or a ated with pre-diabetes, diabetes, or diabetic complication if it labeled anti-glycoprotein or glycolipid antibody. In another decreases sample binding to one or more lectin (for example, embodiment, the detector reagent includes a lectin binding AAL, DSL, PHA-E, ConA, SNA, LEL, or a combination of protein conjugated to a colored latex particle and an addi two or more thereof) by at least about 10% (such as about tional binding partner (such as biotin or BSA-digoxigenin). 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 99%) as compared to a sample from a subject that is not 4. Absorbent Pad treated with the test compound. 0194 The use of an absorbent pad 18 in a lateral flow 0.198. In embodiments, a “compound' or “test compound device is optional. The absorbent pad acts to increase the total may be any Substance or any combination of Substances that volume of sample that enters the device. This increased vol is useful for achieving an end or result. The compounds ume may be useful, for example, to wash away unbound identified using the methods disclosed herein may be of use analyte from the membrane. Any of a variety of materials is for selectively altering a glycosylation profile associated with useful to prepare an absorbent pad, see, for example, Table 2. pre-diabetes or diabetes. Any compound that has potential In some device embodiments, an absorbent pad may be paper (whether or not ultimately realized) to affect glycosylation (i.e., cellulosic fibers). One of skill in the art may select a profile may be tested using the methods of this disclosure. paper absorbent pad on the basis of for example, its thick 0199. Appropriate compounds may be contained in librar ness, compressibility, manufacturability, and uniformity of ies, for example, synthetic or natural compounds in a combi bed volume. The volume uptake of an absorbent made may be natorial library. Numerous libraries are commercially avail adjusted by changing the dimensions (usually the length) of able or may be readily produced; means for random and an absorbent pad. directed synthesis of a wide variety of organic compounds IX. Methods for Identifying Compounds that Selectively and biomolecules, including expression of randomized oli Block or Alter a Pre-Diabetic or Diabetic Glycosylation Pro gonucleotides, such as antisense oligonucleotides and oli file gopeptides, also are known. Alternatively, libraries of natural (0195 The identification herein of altered glycosylation compounds in the form of bacterial, fungal, plant and animal profiles indicative of pre-diabetes, diabetes, and diabetic extracts are available or may be readily produced. Addition complications provides an opportunity to identify com ally, natural or synthetically produced libraries and com pounds that prevent such changes in glycosylation profile. pounds are readily modified through conventional chemical, Disclosed herein are methods for identifying a compound that physical and biochemical means, and may be used to produce selectively decreases or blocks one or more type of glycosy combinatorial libraries. Such libraries are useful for the lation that is increased in pre-diabetes, diabetes, or diabetic screening of a large number of different compounds. complication, Such as one or more type of glycosylation that 0200 Additional test compounds include lectins (for binds to lectins, such as AAL, DSL, PHA-E, ConA, SNA, example, AAL, DSL, PHA-E, ConA, SNA LEL, MAL, or LEL, or a combination of two or more thereof. ECL), glycosylation inhibitors (for example, N-butyldeox 0196. In some examples, the methods include identifying ynojirimycin, castanospermine, 1-deoxymannojirimycin, a compound that selectively binds to one or more type of 1-deoxynoririmycin, Swainsonine, tunicamycin), foods glycosylation that is increased in pre-diabetes, diabetes, or (such as foods containing lectins, for example legumes, such diabetic complication. In a particular example, a sample with as red kidney beans, soybeans or peanuts), and food extracts a glycosylation profile indicative of pre-diabetes, diabetes (such as fruit or vegetable extracts). (such as a urine or saliva sample from a subject having pre 0201 In embodiments, compounds that may alter a gly diabetes or diabetes) is contacted with a test compound. The cosylation profile. Such as a compound that selectively sample binding to one or more lectin indicative of pre-diabe decreases or blocks one or more type of glycosylation that is tes or diabetes (such as AAL, DSL, PHA-E, ConA, SNA, increased in pre-diabetes, diabetes, or diabetic complication, LEL, or a combination of two or more thereof) is determined. may be administered in any Suitable manner, preferably with A decrease in Sample binding to one or more lectin (such as at pharmaceutically acceptable carriers. Pharmaceutically least a 10% decrease, for example 10%, 20%, 30%, 40%, acceptable carriers are determined in part by the particular 50%. 60%, 70%, 80% 90%, or 95% decrease) as compared to composition being administered, as well as by the particular a control or reference indicates a compound that selectively method used to administer the composition. Accordingly, binds to one or more type of glycosylation that is increased in there is a wide variety of suitable formulations of pharmaceu pre-diabetes, diabetes, or diabetic complication. tical compositions of the present disclosure. The pharmaceu 0197) In some examples, a test compound may be admin tically acceptable carriers useful in this disclosure are con istered to a test Subject, such as a Subject with pre-diabetes or ventional. Remington's Pharmaceutical Sciences, by E. W. diabetes (such as an animal model, for example, a mouse or Martin, Mack Publishing Co., Easton, Pa., 15th Edition rat model of diabetes) for a period of time sufficient to (1975), describes compositions and formulations suitable for decrease or block one or more type of glycosylation that is pharmaceutical delivery of the compounds herein disclosed. increased in pre-diabetes, diabetes, or diabetic complication. 0202 Preparations for parenteral administration include One of skill in the art may determine the appropriate time sterile aqueous or non-aqueous solutions, Suspensions, and period of treatment for a particular compound. In general, the emulsions. Examples of non-aqueous solvents are propylene compound is administered to the Subject for at least about one glycol, polyethylene glycol, vegetable oils such as olive oil, day, two days, five days, one week, two weeks, one month, and injectable organic esters such as ethyl oleate. Aqueous US 2012/0208218 A1 Aug. 16, 2012

carriers include water, alcoholic/aqueous Solutions, emul tein spots was performed using Phoretix 2D evolution, ver sions or Suspensions, including saline and buffered media. sion 2005 (Non-Linear Dynamics Ltd). Parenteral vehicles include sodium chloride solution, Ring er's dextrose, dextrose and sodium chloride, lactated Ring Results er's, or fixed oils. Intravenous vehicles include fluid and nutri 0210. Two-dimensional difference gel electrophoresis ent replenishers, electrolyte replenishers (such as those based (2D DIGE) analysis was used to analyze serum glycoproteins on Ringer's dextrose), and the like. Preservatives and other in samples from patients with IGT (FIG. 2A), IFG (FIG. 2B), additives may also be present such as, for example, antimi and T2DM at diagnosis (FIG. 2C) and T2DM after treatment crobials, anti-oxidants, chelating agents, and inert gases and (FIG. 2D). Equal quantities of labeled proteins were labeled the like. and subjected to 2D DIGE. Distinct changes in the serum 0203 Formulations for topical administration may glycoproteome profile were observed in the samples. Some include ointments, lotions, creams, gels, drops, Suppositories, glycoprotein spots were increased in IGT. IFG, or T2DM sprays, liquids and powders. Conventional pharmaceutical samples compared to controls, while other glycoprotein spots carriers, aqueous, powder or oily bases, thickeners and the were decreased in IGT. IFG, or T2DM samples compared to like may be necessary or desirable. controls (FIG. 2). 0204 Compositions for oral administration may include 0211 Salivary glycoproteins were also analyzed by 2D powders or granules, Suspensions or solutions in water or DIGE in samples from patients with IGT (FIG. 3A), IFG non-aqueous media, capsules, Sachets, or tablets. Thickeners, (FIG. 3B), T2DM (FIG. 3C), and T1DM (FIG. 3D). As with flavorings, diluents, emulsifiers, dispersing aids or binders the serum samples, distinct changes in the salivary glycopro may be desirable. teome profile were observed in the samples. Some glycopro 0205 The present disclosure is illustrated by the following tein spots were increased in IGT. IFG, or T2DM samples non-limiting Examples. compared to controls, while other glycoprotein spots were decreased in IGT. IFG, or T2DM samples compared to con EXAMPLES trols (FIG. 3). In addition, the analysis showed that some Example 1 glycoproteins increased in T1DM as compared to T2DM (FIG. 3). Glycoproteome Profile in Pre-Diabetes and Diabetes Example 2 0206. This example describes serum and salivary glyco protein profiles in control, pre-diabetic, and diabetic Subjects. Urinary Glycosylation Profile in Pre-Diabetes and Diabetes Methods 0212. This example describes the glycosylation profile of 0207 Total glycoproteins were enriched from 3 mg of urine samples from non-diabetic and pre-diabetic individu serum or saliva proteins, using Q-proteome glycoprotein als. fractionation columns (Qiagen, Valencia, Calif.) made of lec tins such as ConA, LCH, WGA, and AIL, with standard Methods buffers as provided by the manufacturer. The quantities of the recovered glycoproteins were estimated with a Bio-Rad DC 0213 Urine samples were resuspended in 100 mM car protein assay (Bio-Rad Laboratories, Hercules, Calif.). bonate-bicarbonate buffer, pH 9.6, coated onto ReactibindTM Samples from five subjects in each representative group were plates (Pierce, Rockford, Ill.), and incubated overnight at 4 pooled for purification and gel electrophoresis. C. On the following day, plates were washed in phosphate 0208 Serum or saliva proteins (50 ug) were labeled with buffered saline/Tween (PBST) using a Tecan R) microplate CyDye DIGE Fluor minimal dye (Amersham Biosciences, washer and incubated with either biotinylated AAL, Con A, Piscataway, N.J.) at a concentration of 100-400 pmol of dye? DSL, ECL, GSL-2, HHL, LEL, LTL, MAL, PHA-E, SNA, or 50 ug of protein. Samples were labeled with Cy3 (patient), VVL (Vector Labs, Burlingame, Calif.) diluted in phosphate Cy5 (control), or Cy2 (reference, patient+control), and all buffered saline (PBS). Plates were washed with 1.65 ml three labeled samples were multiplexed and resolved in one PBST and incubated with streptavidin-horseradish peroxi gel. Labeled proteins were purified by acetone precipitation, dase (HRP) (Pierce, Rockford, Ill.) dissolved in PBS. Plates dissolved in IEF buffer and rehydrated onto a 24 or 13-cm were developed with 3,3',5.5' tetramethylbenzidine (TMB) IPG strip (pH 4-7) for 12 hours at room temperature. The IPG Substrate (Neogen Corporation, Lexington, Ky.) and strip was subjected to 1-dimensional electrophoresis at 65-70 quenched with 2NHSO. Plates were analyzed using a Spec kVhrs. The IPG strip was then equilibrated with DTT equili tramax(R) plus microplate reader (Molecular Devices Corpo bration buffer and IAA equilibration buffer for 15 minutes ration, Sunnyvale, Calif.). sequentially before second-dimensional SDS-PAGE analy sis. The IPG strip was then loaded onto an 8-16% SDS-PAGE Results gel and electrophoresis conducted at 80-90 V for 18 hours to 0214. A direct lectin ELISA platform was developed to resolve proteins in the second dimension. determine changes in specific glycosylation profiles in urine 0209 Gels were scanned in a Typhoon 9400 scanner (Am from individuals with pre-diabetes and diabetes. In this plat ersham BioSciences) using appropriate lasers and filters with form, urine samples were coated onto an ELISA plate, and PMTVoltage between 550-600. Images in different channels global changes in particular types of glycosylation were (control vs. IFG, IGT, and T2 DM) were overlaid using assessed by probing with biotinylated plant lectins. In the pseudo-colors, and differences were visualized using current study, a screen of urine was performed using lectins. ImageOuant Software (Amersham Biosciences). 2D-gel Preliminary direct lectin ELISAs were conducted on pooled image analysis to determine the differentially abundant pro urine samples from control, pre-diabetes and diabetes indi US 2012/0208218 A1 Aug. 16, 2012 22 viduals. Relatively weak signals were observed for GSL-2 significantly increased in diabetes samples compared to con (GlcNAc-GlcNAc), HHL (polymannose), LTL (C-12 trols. The ratio of ConA binding to AAL binding in both fucose), and VVL (O-linked GalNac). Strong signals were pre-diabetes and diabetes samples was increased compared to observed for lectins that recognized lactosamine (Gal-B1.4 controls, although this did not reach statistical significance. GlcNAc), C-1.3 or C-1,6 fucosylation, and terminal sialyla tion. TABLE 4 0215. A panel of urines from control subjects and patients with pre-diabetes or diabetes was probed. Following normal Lectin binding ratio in control, pre-diabetic and diabetic urine samples ization by protein concentration, a significantly greater amount of lectin binding was detected in both pre-diabetes Study Group (n and diabetes samples compared to controls using AAL, DSL, Control Pre-diabetes Diabetes and PHA-E biotinylated lectins (Table 3 and FIG. 4). A sig Lectin Ratio (43–50) (44-57) (43-47) nificantly greater amount of binding by ConA and SNA was SNAMAL also observed in diabetes samples compared to controls (Table 3). The other lectins employed did not detect signifi Geometric mean (sc) 1.4 (25) 3.3 (56) 18.9 (7) cant differences between control, pre-diabetic, and diabetic p-value' Referent 0.77 O.OO1 urine samples. However, increased binding by ConA, SNA, ConAMAL and LEL was observed in pre-diabetes samples compared to Geometric mean (sc) 1.8 (54) 11.2 (49) 29.3 (20) controls, and increased LEL binding was observed in diabetes p-value' Referent O.17 O.OO3 samples compared to controls, although these increases were ConAAAL not statistically significant. Geometric mean (sd) 17.9 (66) 89 (11) 92 (10) p-value' Referent O.09 O.09 TABLE 3 Linear regression using GeneralizedLinear Models and Dunnett's T3 post-hoc correction Lectin binding to control, pre-diabetic and diabetic urine samples factor to control for unequal variance across study groups Study Group (n 0217. These data provide a glycosylation profile indica tive of pre-diabetes. The profile includes significantly Control Pre-diabetes Diabetes increased binding of the sample to the lectins AAL, DSL, Lectin (arbitrary units) (44-50) (49-57) (43-47) PHA-E, or a combination of two or more thereof. In contrast AAL the binding of the sample to ConA, SNA, LEL, MAL, and ECL was not significantly changed compared to the control. Geometric mean (sc) 473 (3) 1011 (3) 1063 (3) p-value' Referen O.OO2 O.OO1 Thus, a profile for identifying pre-diabetes may include DSL increased binding of the sample to AAL, DSL, PHA-E or a combination of two or more thereof. The profile may also be Geometric mean (sd)? 472 (3) 1085 (3) 1080 (3) p-value' Referen O.OO3 O.OOS expressed as a ranking by amount of lectin binding using the PHA-E results of a statistical test, Such as in increasing order of p-value. Such a glycosylation profile in pre-diabetes may Geometric mean (sc) 2462 (4) 35118 (7) 38228 (7) p-value' Referen O.O2 O.O2 include increased binding to AALDDSLDPHA ConA E>LELDConA>SNADECL>MAL. A glycosylation profile indicative of pre-diabetes may also include increased lectin Geometric mean (sc) 3507 (59) 20231 (10) 53142 (11) p-value Referen O.24 O.O2 binding ratios, for example expressed as a ranking of lectin SNA binding ratio in increasing order of p-value. Such a glycosy lation profile in pre-diabetes may include increased ConA/ Geometric mean (sc) 2748 (18) 6150 (9) 26680 (3) p-value Referen O.81 O.04 AALDConA/MAL>SNA/MAL. LEL 0218. These data also provide a glycosylation profile indicative of diabetes. The profile may include increased Geometric mean (sc) 2812 (4) 7328 (7) 7910 (6) p-value Referen O.13 O.O7 binding of the sample to the lectins AAL, DSL, PHA-E, MAL ConA, SNA, or a combination of two or more thereof. In contrast, the binding of the sample to LEL, MAL, and ECL Geometric mean (sc) 2171 (3) 1742 (20) 3268 (7) p-value Referen O.94 O.S6 was not significantly changed compared to the control. Thus, ECL a profile for identifying diabetes may include increased bind ing of the sample to AAL, DSL, PHA-E, ConA, SNA, or a Geometric mean (sc) 3930 (3) 5225 (9) 5738 (9) combination of two or more thereof. The profile may also be p-value Referen O.89 0.72 expressed as a ranking by amount of lectin binding using the Linear regression using Generalized Linear Models and Dunnett's 2-sided post-hoc cor section factor for multiple comparisons versus the control results of a statistical test, Such as in increasing order of Lectin concentration 1000 p-value. Such a glycosylation profile in diabetes may include Linear regression using GeneralizedLinear Models and Dunnett's T3 post-hoc correction increased binding tO AAL-DSL-PHA factor to control for unequal variance across study groups E=ConA>SNADLELDECL>MAL. A glycosylation profile 0216 Lectin binding by control, pre-diabetic, and diabetic indicative of diabetes may also include increased lectin bind urine samples was also analyzed as a ratio of lectin binding ing ratios, such as increased SNA/MAL, ConA/MAL, or a (Table 4). The ratio of SNA binding to MAL binding was combination thereof. The glycosylation profile of diabetes significantly increased in diabetes samples compared to con using lectin binding ratios may also be expressed using the trols. The ratio of ConA binding to MAL binding was also results of a statistical test, Such as in increasing order of US 2012/0208218 A1 Aug. 16, 2012 p-value. Such a glycosylation profile in diabetes may include with 100 mM sodium metaperiodate, 50 mM citric acid, pH4 increased SNA/MAL>ConA/MAL>ConA/AAL ratios. for 15 minutes. Urine samples were added to the plate and incubated for 3 hours at room temperature. Following wash Example 3 ing with PBST, biotin-conjugated lectin was added and the Glycosylation of Alpha-1 Acid Glycoprotein in Pre plate was incubated at room temperature for 2 hours. Devel Diabetes and Diabetes opment of the plate was also conducted as described in Example 1. 0219. This example describes the glycosylation pattern (amount and type of glycosylation) of a specific urinary pro tein in individuals with pre-diabetes and diabetes. Results 0222. It was first determined by protein ELISA that A1AG Methods was present in urine in all the pre-diabetic and diabetic con 0220 Concentrations of A1AG in urine were estimated by ditions. The amount of A1AG in the pre-diabetic IGT and sandwich ELISA (Clark and Adams, J. Gen. Virol. 34:475 diabetic (NDM) urine was significantly higher than in con 483, 1977; Nerurkar et al., J. Clin. Microbiol. 20:109-114, trols or IFG (Table 5). 1984). A1AG polyclonal antibody (Dako, Carpinteria, Calif.; 0223) The specific types of glycosylation of A1AG were cat #A0011) was prepared in 100 mM carbonate-bicarbonate probed with lectins by capturing Al AG on the assay plate and buffer, pH 9.6, at a concentration of 2.0 pg/ml and coated on probing the respective Sugars with biotinylated lectins (anti a ReactibindTM plate by incubating overnight at 4°C. The body-lectin ELISA: Table 5). A marginal increase in AAL plate was then washed with PBST and blocked with 3% reactivity on A1AG in urine from IGT subjects was observed. bovine serum albumin (BSA) in PBS for 1.5 hours at room There was a significant increase in the ConA reactivity of temperature. After washing the plates with PBST, appropriate A1AG in IGTurine compared to control. Lastly, there was a dilutions of pure A1AG protein standard (Sigma, St. Louis, significant difference in the amount of SNA-reactive A1AG in Mo.) and urine samples were added to the plate in triplicate NDM urine. Increases in Con A reactivity reflect a greater and incubated for 1 hour. The plates were incubated with amount of biantennary content of A1AG, and increases in appropriate dilution of biotin-conjugated A1AG Dako poly AAL reactivity indicate a greater amount of terminal fucose.

TABLE 5 Al AG protein and lectin binding to Al AG protein from urine samples

Control IFG IGT NDM (n = 42) (n = 16) (n = 27) (n = 24) IGT vs. control NDM vs. control Analyte Mean SD Mean SD Mean SD Mean SD p-value AUROC p-value AUROC A1AG 20.40 6.12 32.32 S.17 54.71 - 4.69 64.70 - 2.12 O.O379 O.669 O.O103 0.723 A1AG + 325.47 6.67 597.119.47 855.92 4.77 331.945.71 O.O4S5 O.612 O.9663 O.S28 ConA A1AG + 678.81 - 2.84 631419.29 777.09 8.24 51O.S7 2.92 O.7242 O.S47 O.2916 O.S90 DSL A1AG + 445.96 - 2.72 604.07 2.21 769.7S 3.25 S34.77 2.76 O.OS66 O.613 O.4769 O.S26 AAL A1AG + 80.1359.91 255.3650.44 440.01 - 39.64 1429.25 - 13.28. O.1OOO O.626 O.OO88 O.743 SNA A1AG + 440.6O7.40 880.61 - 3.85 977.37 S.09 283.06 10.04 O.1099 0.567 0.4144 O.S89 MAL clonal secondary antibody for 1 hour, and washed with 1.65 0224. These data provide glycosylation patterns of A1AG ml of PBST. Streptavidin-HRP conjugate was added at a in pre-diabetes and diabetes. A glycosylation pattern of concentration of 0.1 mg/ml and incubated for 45 min prior to Al AG in pre-diabetes may include increased binding of washing with PBST. TMB liquid substrate was then added A1AG to the lectins ConA, AAL, or a combination thereof. A followed by incubation at room temperature for 5-15 min for glycosylation pattern of MAG in diabetes may include color development. The reaction was stopped by the addition increased binding of A1AG to the lectin SNA. The A1AG glycosylation pattern may also be expressed as a ranking by of 100 ul of 2N HSO4. Absorbance at 450 nm was measured amount of lectin binding using the results of a statistical test, on a Spectramax(R Plus microplate reader (Molecular Such as in increasing order of p-value. Such an A1AG glyco Devices Corporation, Sunnyvale, Calif.). A standard curve Sylation pattern in pre-diabetes may include binding to was generated by either log-log or four-parameter curve fit ConA>AAL>MAL>SNA>DSL. An A1AG glycosylation ting using the Softmax(R) Pro V1.11 software (Molecular pattern in diabetes may include increased binding to Devices Corporation, Sunnyvale, Calif.). The concentrations SNA-DSLDMALDAALDConA. of the individual samples were estimated from the average values of triplicates, in comparison to the standard curve. Example 4 0221 A1AG glycosylation pattern was determined using Glycosylation Profile in Diabetic Complications an antibody-lectin ELISA. A1AG polyclonal antibody was 0225. This example describes the glycosylation profile of coated onto a ReactibindTM plate as described above. Follow urine samples from individuals with diabetic complications, ing blocking with 3% BSA in PBS, the plates were treated Such as chronic renal failure. US 2012/0208218 A1 Aug. 16, 2012 24

Methods Example 5 0226. The glycosylation profile of urinary proteins was Exemplary Diagnostic Study determined by lectin ELISA, as described in Example 2. Concentrations of A1AG and A1AT in urine were estimated 0229. In embodiments, a saliva or urine sample may be by sandwich ELISA as described in Example 3. obtained from Subjects suspected to have pre-diabetes (such as having an OGTT two-hour plasma glucose of greater than Results or equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM) and/or a fasting plasma glucose (FPG) of greater than 0227. A panel of urines from control subjects and patients 100 mg/dL and less than 126 mg/dL (5.6-6.9 mM)). A lectin with T2DMorT2DM with chronic renal failure (T2DM/renal ELISA may be performed on the sample, and a glycosylation failure) were probed. The amount of both A1AG and A1AT in profile (as defined by binding to one or more particular lec both T2DM and T2DM/renal failure urine was significantly tins, such as PHA-E, LEL, DSL, AAL, SNA, ConA, or MAL) higher than in controls (Table 6). A significantly greater may be determined relative to a glycosylation profile in a amount of lectin binding was detected in both T2DM and sample from a control subject without diabetes. A protein T2DM/renal failure samples compared to controls using AAL ELISA may be performed on the sample, and the amount of biotinylated lectin (Table 6). T2DM/renal failure samples A1AG and/or A1AT may be determined relative to the also had significantly increased SNA binding compared to amount of these proteins in a sample from the control. controls; however, T2DM samples did not have increased 0230. An analysis may be performed, and the glycosyla SNA binding compared to controls (Table 6). ConA did not tion profile may be altered compared to the glycosylation detect significant differences between control, T2DM, and profile of the sample from the normal Subject. An increase in T2DM/renal failure urine samples. The amount of A1AG and one or more types of glycosylation in the sample from the A1AT protein, as well as AAL and SNA reactivity was also Subject may identify the Subject as pre-diabetic. The glyco higher in T2DM/renal failure samples than in T2DM Sylation profile may also be determined relative to the glyco samples. Sylation profile in a sample from a subject known to be dia betic. An analysis may be performed, and the glycosylation TABLE 6 profile may be altered compared to the glycosylation profile of the sample from the diabetic subject. This may confirm that Lectin binding and protein expression in T2DM and renal failure the subject is pre-diabetic. Control T2DM Renal 0231. An analysis may be performed, and the amount of (n = 10)* T2DM (n = 20)* Failure (n = 9)* A1AG and/or A1AT may be altered compared to the amount A1AG of A1AG and/or A1AT in the sample from the normal subject. An increase in the amount of A1AG and/or A1AT in the Geometric Mean (SD) 24 (2) 137 (8) 7280 (2) sample from the Subject may identify the Subject as pre P value Referen O.O1 <0.001 A1AT diabetic. The amount of A1AG and/or A1AT may also be determined relative to the amount of A1AG and/or A1AT in a Geometric Mean (SD) 7 (3) 109 (11) 7500 (4) sample from a Subject known to be diabetic. An analysis may P value Referen O.OO2 <0.001 be performed, and the amount of A1AG and/or A1AT is AAL altered compared to the amount of A1AG and/or A1AT in the Geometric Mean (SD) 376 (2) 774 (2) 9197 (2) sample from the diabetic subject. This may confirm that the P value? Referen O.O3 <0.001 Subject is pre-diabetic. SNA Geometric Mean (SD) 39 (2) 70 (3) 2295 (3) Example 6 P value Referen O.17 <0.001 ConA Exemplary Lateral Flow Device Diagnostic Test Geometric Mean (SD) 234 (7) 93 (4) 1570 (3) 0232 FIG. 5 schematically illustrates an exemplary lateral P value Referen O.61 O.13 flow device for diagnosis of pre-diabetes or diabetes, includ ing a test line for pre-diabetes or diabetes and a reference line. *Protein (ngi ml) Multiple comparisons against the control using Dunnett's T3 post-hoc correction factor to The device may detect binding of glycoproteins in a sample to account for unequal variance specific glycoprotein-binding molecules using a competitive ’2-sided Dunnett's post-hoc correction factor versus the control Concentration/1 0,000 assay format including a test lectin (TL) and a reference lectin (RL). 0228. These data provide glycosylation profiles for diabe 0233. In the exemplary device, the conjugate pad 19 may tes and the diabetic complication of renal failure. A glycosy include a test lectin binding protein (TLBP) conjugate 40 lation profile in diabetes includes increased binding of the (such as the TLBP fetuin covalently attached to blue latex sample to the lectin AAL. A glycosylation profile of a diabetic particles and biotin) and a reference lectin binding protein complication may include increased binding of the sample to (RLBP) conjugate 42 (such as a protein fraction that is affinity the lectins AAL and SNA, or a combination thereof. The absorbed for TL binding and affinity purified for RL binding glycosylation pattern may also be expressed as a ranking by covalently attached to blue latex and BSA-digoxigenin). The amount of lectin binding using the results of a statistical test, TL (such as AAL) may be immobilized on the nitrocellulose Such as in increasing order of p-value. Such glycosylation membrane distal to the conjugate pad at the TL capture line profile in diabetes may include binding to 44. The RL (such as MAL) may be immobilized on the AALDSNA>ConA. A glycosylation profile in diabetic com nitrocellulose membrane distal to the TL test line 20 at the RL plication may include increased binding to capture line 46. In embodiments, the TL test line 20 may be AAL=SNA>ConA. streptavidin and the RL test line 22 may be anti-digoxigenin. US 2012/0208218 A1 Aug. 16, 2012

0234. The test may be performed by applying a sample 5. The lectin binding test device of claim 1, wherein the test (such as urine or saliva) from a Subject to the sample appli device provides a quantitative measure of binding for each of cation pad 14. The sample may flow through the conjugate the first and second lectins. pad 19, releasing the TLBP conjugate and the RLBP conju 6. The lectin binding test device of claim 5, wherein the test gate. The displaced TLBP conjugate may be captured by the device provides a ratio of binding for each of the first and TL test line 20. The displaced RLBP conjugate may be cap second lectins. tured by the RL test line 22. The TL test line intensity may be compared to the RL test line intensity (for example, visually 7. The lectin binding test device of claim 6, wherein the first or using a reader). If the TL test line intensity is greater than and second lectins comprise one pair selected from Sambucus the RL test line intensity, then the Subject may be diagnosed nigra lectin and Maackia amurensis lectin I, Concanavalin A as normal. If the TL test line intensity is less than the RL test and Maackia amurensis lectin I, and Concanavalin A and line intensity, then the Subject may be diagnosed as having Aleuria aurantia lectin. pre-diabetes or diabetes. 8. The lectin binding test device of claim 7, wherein an 0235 Although certain embodiments have been illus increase in a ratio of binding of Sambucus migra lectin to trated and described herein, it will be appreciated by those of Maackia amurensis lectin I, Concanavalin A to Maackia ordinary skill in the art that a wide variety of alternate and/or amurensis lectin I, or Concanavalin A to Aleuria aurantia equivalent embodiments or implementations calculated to lectin indicates that the sample is from a subject having dia achieve the same purposes may be substituted for the embodi betes. ments shown and described without departing from the scope. 9. The lectin binding test device of claim 1, wherein the Those with skill in the art will readily appreciate that embodi device further comprises one or more specific binding agents ments may be implemented in a very wide variety of ways. for at least one of alpha-1 acid glycoprotein (A1AG) and This application is intended to cover any adaptations or varia alpha-1 antitrypsin (A1AT) protein. tions of the embodiments discussed herein. Therefore, it is 10. The lectin binding test device of claim 9, wherein the manifestly intended that embodiments be limited only by the one or more specific binding agents for of alpha-1 acid gly claims and the equivalents thereof. coprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) protein What is claimed is: provides a quantitative measure of an amount of alpha-1 acid 1. A lectin-binding test device for determining a glycosy glycoprotein (A1AG) and/or alpha-1 antitrypsin (A1AT) pro lation profile in a sample from a Subject, comprising: tein in the sample. a first lectin; 11. The lectin binding test device of claim 10, wherein an a second lectin; increase in the quantity of binding of A1AG and/or A1AT as a Support for the first and second lectins; and compared to a control value indicates that the Subject has an indicator to detect binding of the first and second lectins pre-diabetes or diabetes. to a glycosylated protein in the sample, and to provide an indication of the glycosylation profile of the sample. 12. The lectin binding test device of claim 1, wherein the 2. The lectinbinding test device of claim 1, wherein the first first and/or second lectins bind alpha-1 acid glycoprotein. and second lectins comprise Aleuria aurantia lectin, Con 13. The lectin binding test device of claim 1, wherein the canavalin A. Datura stramonium lectin, Erythrina Cristagalli first and second lectins comprise Sambucus migra lectin and lectin, Lycopersicon esculentum lectin, Maackia amurensis Maackia amurensis lectin I, and wherein an increase in a ratio lectin I, Phaseolus vulgaris agglutinin, and/or Sambucus of binding of Sambucus nigra lectin to Maackia amurensis nigra lectin. lectin I indicates that the sample is from a subject having 3. The lectinbinding test device of claim 1, wherein the first diabetes. and second lectins comprise one pair selected from Aleuria 14. The lectin binding test device of claim 1, wherein the aurantia lectin, Datura Stramonium lectin, and Phaseolus first and second lectins comprise Concanavalin A and vulgaris agglutinin, and wherein increased lectin binding by Maackia amurensis lectin I, and wherein an increase in a ratio Aleuria aurantia lectin, Datura Stramonium lectin, and/or of binding of Concanavalin A to Maackia amurensis lectin Phaseolus vulgaris agglutinin indicates that the sample is indicates that the sample is from a Subject having diabetes. from a subject having pre-diabetes. 15. The lectin binding test device of claim 1, wherein the 4. The lectinbinding test device of claim 1, wherein the first first and second lectins comprise Concanavalin A and Aleu and second lectins comprise one pair selected from Con ria aurantia lectin, and wherein an increase in a ratio of canavalin A and/or Sambucus nigra lectin, and wherein binding of Concanavalin A to Aleuria aurantia lectin indi increased lectin binding by Concanavalin A and/or Sambu cates that the sample is from a subject having diabetes. cus nigra lectin indicates that the sample is from a subject having diabetes. c c c c c