US20050142618A1

US20050142618A1 - Method for diagnosing diseases based on levels of anti-glycan antibodies

Info

Publication number: US20050142618A1
Application number: US10/843,033
Authority: US
Inventors: Nir Dotan; Avinoam Dukler
Original assignee: Glycominds Ltd
Current assignee: Glycominds Ltd
Priority date: 2003-12-03
Filing date: 2004-05-11
Publication date: 2005-06-30
Also published as: ES2349804T3; US7592150B2; US20050124006A1; US20100099124A1; DE602004027529D1; IL175692A0; ATE470152T1

Abstract

Disclosed are methods for diagnosing Crohn's disease (CD) or anti-phospholipid Syndrome (APS) by measuring levels of antibodies to glycans in a biological sample.

Description

RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S. Ser. No. 10/728,227, filed Dec. 3, 2003. The contents of this application are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to a method for diagnosing diseases by detecting levels of antibodies to glycans in a subject. More particularly, the invention relates to methods for diagnosing digestive diseases such as Crohn's disease (CD), or anti-phospholipid syndrome (APS).

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD), which occurs world-wide and afflicts millions of people, is the collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC). IBD and irritable bowel syndrome (IBS) will affect one-half of all Americans during their lifetime, at a cost of several billion dollars. A primary determinant of these high medical costs is the difficulty of diagnosing digestive diseases. The cost associated with IBD and IBS is compounded by lost productivity, with persons suffering from these disorders missing an average of at least eight more days of work annually than persons not suffering from these disorders.
Symptoms associated with Crohn's disease include, e.g., abdominal pain, chronic diarrhea, rectal bleeding, weight loss and cramping. These symptoms are also found in irritable bowel syndrome and other inflammatory bowel diseases. This makes definitive diagnosis of CD extremely difficult. In fact, only about one-tenth of the several million people suspected of suffering from CD are actually diagnosed with the disease The difficulty in differentially diagnosing CD from other digestive diseases like UC and IBS hampers early and effective treatment of these diseases.
Crohn's disease (ileitis regionalis or ileitis terminalis) may affect any part of the gut with the ileum and colon as the most commonly affected sites. In CD the inflammation is asymmetrical and segmental, with areas of both healthy and diseased tissue. By contrast, ulcerative colitis (hemorrhagic idiopathic proctocolitis) is characterized by symmetrical inflammation—restricted to mucosa and submucosa—ascending uninterrupted from rectum to colon.
Crohn's disease is typically diagnosed using upper or lower GI endoscopy and/or by X-ray examination of the small intestine including ileum. In CD no typical endoscopic picture is shown, while in UC the typical pattern detected is an inflamed red mucosa with bleeding. In CD biopsy specimens reveal transmural inflammation with lymphocytes, macrophages and plasma cells while mucosal/submucosal inflammation with granulocytes, eosinophiles and plasma cells are typical findings in UC. When inflammation is limited to the colon, it can be very difficult to differentiate between UC or colon-restricted UC (which also known as CD colitis).
Antiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis, recurrent miscarriages, and thrombocytopenia, which is a low number of blood platelets that can lead to bleeding, seen as bruising and tiny red dots on the skin. Patients with APS also may experience symptoms of stroke such as transient ischemic attacks (TIAs).
Antiphospholipid syndrome is typically diagnosed based on these clinical manifestations and on laboratory test results. A blood sample is analyzed for the presence of antibodies that react with naturally occurring proteins complexed with phospholipids. These are called antiphospholipid antibodies or anticardiolipin antibodies (cardiolipin is one type of phospholipid used in lab tests). Sometimes these antibodies are called lupus anticoagulants when clotting assays are used for their detection.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that patients with Crohn's disease (CD) or anti-phospholipid syndrome (APS) have elevated serum levels of certain IgG, IgA, and IgM isotype antibodies specific for certain glycan structures, as compared to as compared to the serum levels of these antibodies in healthy individuals or in individuals with other types of gastrointestinal diseases.
Among the advantages of the invention is a highly sensitive serological testing method for definitively distinguishing CD from other digestive diseases, even when the inflammation is limited to the colon only. The highly sensitive primary screening assays according to the invention provide physicians with an inexpensive assay for rapidly distinguishing individuals with CD from non diseased individuals, or individuals having UC or IBS. This facilitates earlier and more appropriate therapeutic intervention and minimizing uncertainty for patients and their families.
In one aspect, the invention provides a method of diagnosing Crohn's disease in a subject by providing a test sample from the subject and detecting in the test sample at least one of the following anti-glycan antibodies: an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)G(cNAc(β) antibody, an anti-Gal 3-sulphate (β) antibody, an anti-GlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-Gal(α) antibody, an anti-Gal(β) antibody, an anti-GalNAc(α) antibody, an anti-Glc(α) antibody, an anti-Gal(β,1-6)Gal(β) antibody and an anti-GlcNAc(β,1-6)GalNAc(α) antibody. The presence of one or more of the antibodies in the test sample indicates the subject has Crohn's disease.
In some embodiments, levels of the anti-glycan antibody or antibodies in the test sample are compared to the levels of anti-glycan antibodies in a control sample. The control sample is chosen from a group that includes one or more individuals known to have or not to have a gastrointestinal disorder, or to have or not to have a gastrointestinal disorder other than Crohn's disease. When the control sample is from an individual or individuals that do not have Crohn's disease, or has a gastrointestinal disease other than Crohn's disease, elevated levels in the test sample relative to the control sample indicates that the subject has Crohn's disease.
In some embodiments, the control sample is from one or more individuals with a gastrointestinal disorder that is irritable bowel syndrome, ulcerative colitis or other digestive diseases. In some embodiments, the control sample is from one or more individuals that do not have a gastrointestinal disorder.
In various embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of these antibodies are detected.
In some embodiments, the method further includes determining whether the test sample has an anti-Mannan antibody, which is also known as an anti-Saccharomyces cerevisiae antibody (ASCA). The presence of the anti-Mannan antibody in the sample indicates the subject has Crohn's Disease.
In some embodiments, the method further includes determining whether the test sample has an anti-neutrophil cytoplasmic antibodies (ANCA). The presence of ANCA indicates the subject does not have Crohn's Disease but may have Ulcerative Colitis.
The test sample can be, e.g., a biological fluid. Examples of biological fluids include, e.g., whole blood, serum, plasma, spinal cord fluid, urine, or saliva.
In some embodiments, one, two, three, four or all five of an anti-Glc(β,1-3)Glc(β) antibody, anti-Man(α,1-3)Man(α) antibody, anti-Man(α,1-3)[Man(α,1-6)]Man(α) antibodies, anti-Man(α) and/or anti-Mannan antibodies are detected.
The method can optionally include determining the isotype of the antibody. For example the method can include determining whether the antibody is an IgM, IgA, or IgG-type antibody. In some embodiments, the method is used to identify and compare one or more of an anti-Glc(β)IgG antibody, an anti-Glc(β,1-3)Glc(β) IgG antibody, an anti-Glc(β,1-4)Glc(β) IgG antibody, an anti-GlcNAc(β) 6-sulfate IgG antibody, an anti-Man(α) IgG antibody, an anti-Man(α,1-3)[Man (α,1-6)]Man(β) IgG antibody, an anti-Man(α,1-3)Man(α) IgG antibody, an anti-Mannan IgG antibody an anti-Mannan IgA antibody, an anti-Xylan IgG antibody, or an anti-Man(α,1-2)Man(α) IgG antibody.
In some embodiments, a subject is scored as having CD if the test sample has elevated levels of IgG anti-Glc(β,1,1-3)Glc(β), IgG anti-Man(α,1-3)Man(α), IgG anti Mannan (ASCA) antibodies, or IgA anti Mannan (ASCA) antibodies, but does not have elevated levels of ANCA.
In some embodiments, a subject is scored as having IBD if the test sample has elevated levels of IgG anti-Glc(β,1-3)Glc(β), IgG anti anti-Man(α,1-3)Man(α), IgG anti Mannan (ASCA) antibodies, IgA anti Mannan (ASCA) antibodies, or ANCA.
In some embodiments, a subject is scored as having a non-IBD digestive disease if the test sample has elevated levels of IgA anti-GlcNAc(β,1-4)GlcNAc(β), and low levels of anti Mannan (ASCA) antibodies.
In some embodiments, the anti-glycan antibody or antibodies are detected using a fluorescent antibody, or are detected using an enzyme-linked immunoabsorbent assay (ELISA).
The test sample can be, e.g., a biological fluid. Examples of biological fluids include, e.g., whole blood, serum, plasma, spinal cord fluid, urine, or saliva.
The method can optionally include determining the isotype of the antibody. For example the method can include determining whether the antibody is an IgM, IgA, or IgG-type antibody.
In another aspect, the invention provides a method for diagnosing Crohn's disease in a subject. The method includes providing a test sample from a subject and determining whether an anti-glycan antibody is present in the test sample. At least one anti-glycan antibody is an IgG Glc(β,1-3)Glc(β) antibody or an IgG anti-Man(α,1-3)Man(α) antibody. The presence of at least one antibody in the test sample indicates the subject has Crohn's disease.
In some embodiments, levels of the anti-glycan antibody or antibodies in the test sample are compared to the levels of anti-glycan antibodies in a control sample. The control sample is chosen from a group that includes one or more individuals known to have or not to have a gastrointestinal disorder, or to have or not to have a gastrointestinal disorder other than Crohn's disease. When the control sample is from an individual or individuals that do not have Crohn's disease, or has a gastrointestinal disease other than Crohn's disease, elevated levels in the test sample relative to the control sample indicates that the subject has Crohn's disease.
In some embodiments, the control sample is from one or more individuals with a gastrointestinal disorder that is irritable bowel syndrome or ulcerative colitis or other digestive diseases. In some embodiments, the control sample is from one or more individuals that do not have a gastrointestinal disorder.
In a further aspect, the invention provides a method of differentially diagnosing Crohn's disease or inflammatory bowel disease in a subject. The method includes providing a test sample from a subject and determining whether the sample has an antibody that is an anti-neutrophil cytoplasmic antibody (ANCA), an IgG anti-Glc(β,1-3)Glc(β) antibody, an IgG ASCA and/or IgA ASCA. The absence of ANCA and the presence of at least one of the IgG anti-Glc(β,1-3)Glc(β) IgG ASCA, and IgA ASCA antibodies in the test sample indicates the subject has Crohn's disease, and the presence of at least one of the antibodies in the test sample indicates the subject has inflammatory bowel disease (IBD).
In some embodiments, the anti-glycan antibody or antibodies are detected using a fluorescent antibody, or are detected using an enzyme-linked immunoabsorbent assay (ELISA).
The test sample can be, e.g., a biological fluid. Examples of biological fluids include, e.g., whole blood, serum, plasma, spinal cord fluid, urine, or saliva.
The invention additionally provides a method of differentially diagnosing Crohn's disease colitis and ulcerative colitis in a subject. The method includes providing a test sample from a subject and determining levels of at least one an anti-glycan antibody in the sample. The anti-glycan antibody can be one or more of an IgG anti-Gal(α,1-4)GlcNAc(α) antibody, an IgG anti-Gal(β,1-4)GlcNAc(β) antibody, an IgG anti-GalNAc(α) antibody, an IgG anti-Glc(α) antibody, an IgG anti-Glc(β) antibody, an IgG anti-GlcNAc(β,6-Sulphate) antibody, an IgG anti-GlcNAc(β) antibody, an IgG anti-GlcNAc(β,1-6)GalNAc(α) antibody, an IgA anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an IgA anti-Gal(α,1-4)Gal(β,1-4), Glc(β) antibody, an IgA anti-Gal(β) antibody, an IgA anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody, an IgA anti-Gal(β,1-3)GlcNAc(β) antibody, an IgA anti-Gal(β,1-6)Gal(β) antibody, an IgA anti-GalNAc(α) antibody, an IgA anti-GalNAc(β) antibody, IgA an anti-Glc(β) antibody, an IgA anti-Glc(β,1-3)Glc(β) antibody, an IgA anti-GlcNAc(β) antibody, an IgA anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an IgA anti-GlcNAc(β,1-3)GalNAc(α) antibody, an IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an IgA anti-GlcNAc(β,1-6)GalNAc(α) antibody, and an IgA anti-Xyl(β) antibody. The presence of the at least one antibody in the test sample indicates the subject has Crohn's disease colitis.
In some embodiments, the method further includes comparing the levels of the at least one anti-glycan antibody in the test sample to the levels of the at least one anti-glycan antibody in a control sample, wherein the control sample is selected from the group consisting of one or more individuals known to have or not to have Crohn's disease colitis or known to have or not to have ulcerative colitis (UC).
In some embodiments, the method includes determining whether an additional anti-glycan antibody or antibodies are present in the sample. The additional anti-glycan antibody is one or more of an IgG anti-Gal(α) antibody, an IgG anti-Man(α) antibody, an IgG anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an IgG anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an IgG anti-Man(α,1-3)Man(α) antibody, an IgA anti-Man(α) antibody, an IgA anti-Man(α,1-2)Man(α) antibody, an IgA anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an IgA anti-Man(β,1-3)Man(α) antibody, an IgA anti-Man(α,1-6)Man(α) antibody, an IgA anti-Man(β) antibody, and an IgA anti-X(α) antibody. The presence of the additional antibody or antibodies in the test sample indicates the subject has Crohn's disease colitis.
In some embodiments, the additional antibody or antibodies is an IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody and/or and IgG anti-Man(α,1-3)Man(α) antibody.
In some embodiments, the method includes detecting at least two, three, four, five, six seven, eight, nine, ten, eleven or twelve of the antibodies.
In some embodiments, the test sample is a biological fluid (e.g., whole blood, serum, plasma, urine, or saliva).
Also provided by the invention is a method for differentially diagnosing inflammatory bowel disease (IBD) or non-IBD digestive disease (NIC) in a subject. The method includes providing a test sample from a subject with symptoms of NIC or IBD and determining if anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate Antibodies (ACCA) and anti-mannan (ASCA) antibodies are present in the sample. The presence of ACCA antibodies and the absence of ASCA antibodies in the sample indicates the subject has NIC.
In some embodiments, levels of anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate Antibodies (ACCA) and/or anti-mannan (ASCA) antibodies are determined by comparing levels of the antibodies to levels of antibodies in a reference sample from a subject known to have IBD. A higher level of ACCA antibodies and a lower level of ASCA antibodies in the test sample relative to the reference sample indicates the patient has NIC.
In some embodiments, the method further includes determining whether the test sample has anti-laminarobioside (Glc(β,1-3)Glc(β)) Carbohydrate Antibodies (ALCA) antibodies, wherein the absence of ALCA antibodies in the sample indicates the subject has-NIC.
In some embodiments, the anti-glycan antibody or antibodies are detected using a fluorescent antibody, or are detected using an enzyme-linked immunoabsorbent assay (ELISA).
The test sample can be, e.g., a biological fluid. Examples of biological fluids include, e.g., whole blood, serum, plasma, spinal cord fluid, urine, or saliva.
The invention additionally provides reagents for detecting anti-glycan antibodies that reveal the presence of Crohn's Disease. The reagents include one or more carbohydrates that specifically react with an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Mannan antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-Gal 3-sulphate(β) antibody, an anti-aGlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, and/or an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β) antibody. In some embodiments, the reagents are attached to a solid phase.
Also within the invention are arrays that include reagents (preferably carbohydrate reagents) that specifically detect the disease-detecting antibodies disclosed herein. For example, an array useful for detecting CD can include one or more reagents that detect an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(p antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Mannan antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,14)GlcNAc(β) antibody, an anti-Gal 3-sulphate(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, or an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β) antibody.
In some embodiments, the reagents that are used to specifically bind and detect those anti glycans antibodies are the specific glycan structures. In other embodiments, the reagents are other molecules or macromolecules that include the specific glycan structure. For example, the anti-Glc(β,1-3)Glc(β) antibody can be detected using the polysaccharide β-D(1-3) Glucan, a polymer of glucose units connected in a (β,1-3) glycosidic bond. Thus, the glycan itself can be used for detecting the corresponding antibody or antibodies, as can any carbohydrate, peptide, protein, or any other molecular structure that includes the glycan.
In some embodiments, the reagents that are used to specifically bind and detect the anti glycans antibodies of the invention are peptides that mimic the carbohydrate antigens of the invention. The peptides can be used to identify specific anti glycan antibodies.
The array may additionally include a reagent or reagent, e.g., a carbohydrate reagent or reagents, that detect an anti-Mannan (ASCA) antibody or a ANCA.
In some embodiments, the glycans are attached to the array via a linker. A suitable linker includes at least one ethylene glycol derivative, at least two cyanuric chloride derivatives and an anilino group.
In some embodiment, at least two of the reagent or reagents are provided at the same location on the addressable array.
In some embodiments, the array includes a reagent, e.g., a glycan reagent that detects an anti-Glc(β,1-3)Glc(β) antibody and/or an IgG anti-Man(α,1-3)Man(α) antibody.
Other arrays include arrays useful for differentially diagnosing Crohn's disease or inflammatory bowel disease in a subject. The array includes one or more reagents (e.g., glycan or peptide reagents) that detect an anti-neutrophil cytoplasmic antibody (ANCA), an anti-Glc(β,1-3)Glc(β) antibody, an ASCA; or an ASCA. In some embodiments, the array includes, one, two, or three of these reagents.
The invention additionally provides an array of reagents (e.g., glycan or peptide reagents) useful for differentially diagnosing Crohn's disease colitis and ulcerative colitis in a subject. The arrays include one or more reagents that detect an anti-Gal(α,1-4)GlcNAc(α) antibody, an anti-Gal(β,1-4)GlcNAc(β) antibody, an anti-GalNAc(α) antibody, an anti-Glc(α) antibody, an anti-Glc(β) antibody, an anti-GlcNAc(β,6-Sulphate) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β, 1-6)GalNAc(α) antibody, an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-Gal(α,1-4)Gal(β,1-4) Glc(β) antibody, an anti-Gal(β) antibody, an anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody, an anti-Gal(β,1-3)GlcNAc(β) antibody, an anti-Gal(β,1-6)Gal(β) antibody, an anti-GalNAc(α) antibody, an anti-GalNAc(β) antibody, an anti-Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β, 1-3)Gal(β,1-4)Glc(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(α) antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-GlcNAc(β,1-6)GalNAc(α) antibody, and an anti-Xyl(β) antibody. In some embodiments, the array includes reagents that bind 2, 3, 4, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of these antibodies.
The array may additionally include a reagent, e.g, a glycan or peptide reagent, that detects an anti-Gal(α) antibody, an anti-Man(α) antibody, anti-Man(α,1-3)Man(α,1-6)Man(13) antibody, an anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an anti-Man(β,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(β) antibody, and/or an anti-X(α) antibody. In some embodiments, the array includes reagents that bind 2, 3, 4, 6, 6, 7, 8, 9, 10, 11, or 12 of these antibodies.
The array may additionally include a reagent (e.g., a glycan or peptide reagent) that detects an anti-GlcNAc(β,1-4)GlcNAc(β) antibody and/or an anti-Man(α,1-3)Man(α) antibody.
Also provided by the invention is an array useful for differentially diagnosing inflammatory bowel disease (IBD) or non-IBD digestive disease NIC). The array includes a reagent (e.g., a glycan or peptide reagent) that detects anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate antibodies (ACCA) and/or anti-mannan (ASCA) antibodies. The array may optionally include a reagent that detects anti-laminarobioside (Glc(β,1-3)Glc(β)) Carbohydrate Antibodies (ALCA).
The invention additionally provides kits that include reagents for detecting anti-glycan antibodies that reveal the presence of Crohn's Disease. The kits include one or more carbohydrate reagent(s) that specifically reacts with an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfateantibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Mannan antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-Gal 3-sulphate(β) antibody, an anti-aGlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, and/or an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(P antibody. The kits may be provided in one or more containers. In some embodiments, the kits contain directions for using the kits to perform the methods described herein. The kits may optionally include reagents for detecting antibody isotypes (e.g., IgA, IgG, and IgM antibodies).
In some embodiments, the kits include reagents that are used to specifically bind and detect those anti glycans antibodies that are the specific glycan structures. In other embodiments, the reagents in the kits are other molecules or macromolecules that include the specific glycan structure. For example, the anti-Glc(β,1-3)Glc(β) antibody can be detected using the polysaccharide β-D(1-3) Glucan, a polymer of glucose units connected in a (β,1-3) glycosidic bond. Thus, the glycan itself can be used for detecting the corresponding antibody or antibodies, as can any carbohydrate, peptide, protein, or any other molecular structure that includes the glycan.
In some embodiments, the kits include reagents that are used to specifically bind and detect ASCA and/or ANCA.
Also provided by the invention are kits useful for differentially diagnosing Crohn's disease or inflammatory bowel disease in a subject. The kit includes one or more reagents (e.g., glycan or peptide reagents) that detect an anti-neutrophil cytoplasmic antibody (ANCA), an anti-Glc(β,1-3)Glc(β) antibody, an ASCA; or an ASCA. In some embodiments, the kit includes, one, two, or three of these reagents.
The invention additionally provides a kit of reagents (e.g., glycan or peptide reagents) useful for differentially diagnosing Crohn's disease colitis and ulcerative colitis in a subject. The kits include one or more reagents that detect an anti-Gal(α,14)GlcNAc(α) antibody, an anti-Gal(β,1-4)GlcNAc(β) antibody, an anti-GalNAc(α) antibody, an anti-Glc(α) antibody, an anti-Glc(β) antibody, an anti-GlcNAc(β,6-Sulphate) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β,1-6)GalNAc(α) antibody, an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-Gal(α,14)Gal(β,1-4) Glc(β) antibody, an anti-Gal(β) antibody, an anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody, an anti-Gal(β,1-3)GlcNAc(β) antibody, an anti-Gal(β,1-6)Gal(β) antibody, an anti-GaNAc(α) antibody, an anti-GalNAc(β) antibody, an anti-Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(α) antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-GlcNAc(β,1-6)GalNAc(α) antibody, and an anti-Xyl(β) antibody. In some embodiments, the kit includes reagents that bind 2, 3, 4, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of these antibodies.
The kit may additionally include a reagent, e.g, a glycan or peptide reagent, that detects an anti-Gal(α) antibody, an anti-Man(α) antibody, anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α,1-6)Man(β) antibody, an anti-Man(β,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(β) antibody, and/or an anti-X(α) antibody. In some embodiments, the kit includes reagents that bind 2, 3, 4, 6, 6, 7, 8, 9, 10, 11, or 12 of these antibodies.
The kit may additionally include a reagent (e.g., a glycan or peptide reagent) that detects an anti-GlcNAc(β,1-4)GlcNAc(β) antibody and/or an anti-Man(α,1-3)Man(α) antibody.
Also provided by the invention is a kit useful for differentially diagnosing inflammatory bowel disease (IBD) or non-IBD digestive disease (NIC). The kit includes a reagent (e.g., a glycan or peptide reagent) that detects anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate antibodies (ACCA) and/or anti-mannan (ASCA) antibodies. The kit may optionally include a reagent that detects anti-laminarobioside (Glc(β,1-3)Glc(β)) Carbohydrate Antibodies (ALCA).
Also within the invention is a method of diagnosing anti-phospholipid syndrome in a subject by providing a test sample from a subject and detecting in the test sample an anti-chitobiose antibody. Levels of the anti-GlcNAc(β,1-14)GlcNAc(β) antibody in the test sample are compared to the levels of the antibody in a control sample. The control sample is selected from group of one or more individuals known to have or not to have anti-phospholipid syndrome. When the control sample has one or more individuals that to not have APS, an elevated level of anti-GlcNAc(β,1-4)GlcNAc(β) antibodies in the test sample as compared to the control sample indicates the subject has APS.
In some embodiments, the method also includes detecting binding to a β-2 glycoprotein, and comparing the level of binding to the β-2 glycoprotein in the test sample to the level of binding to β-2 glycoprotein in the control sample. Increased binding to the β-2 glycoprotein in the test sample relative to a control sample taken from a non-APS individual or individuals indicates the subject has APS.
The test sample can be, e.g., a biological fluid. Examples of biological fluids include, e.g., whole blood, serum, plasma, spinal cord fluid, urine, or saliva.
The method can optionally include determining the isotype of the antibody. For example the method can include determining whether the antibody is an IgM, IgA, or IgG-type antibody. Also within the invention is an array that includes a reagent (preferably a carbohydrate reagent) that specifically detects and anti-GlcNAc(β,1-4)GlcNAc(β) antibody and (optionally) a reagent that detects a β-2 glycoprotein for detecting APS.
The invention additionally provides kits for diagnosing APS that include reagents for detecting an anti-chitobiose antibody and (optionally) a β-2 glycoprotein. In some embodiments, the kits contain directions for using the kits to perform the methods described herein.
Any of the kits described herein can be provided with instructions for using the kit. In addition, in some embodiments, the kits are provided with reagents that specifically detect an antibody isotype, e.g., the kit may include one, two, or three reagents that that detect IgA, IgG, IgD or IgM antigodies.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patent, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a Receiver Operator Characteristic (ROC) curve for differentiation between individuals with CD and individuals with other digestive diseases for IgGb3 Gb and IgG Mannan antibodies.
FIG. 2A is a box plot graph of the difference between CD colitis and UC groups for the levels of some antiglycan IgG antibodies.
FIG. 2B is a box plot graph of the difference between CD colitis and UC groups for the levels of some antiglycan IgA antibodies.
FIGS. 3A-3C are box plots showing levels of ASCA (IgG), ALCA (IgG) and ACCA (IgA) in CD, UC, and NIC patients.
FIG. 4A is a Receiver Operator Characteristic (ROC) curve of IgA ACCA (GNb4GNb), IgG ALCA (Gb3Gb) and IgG ASCA (Mannan) for differentiating between CD and UC.
FIG. 4B is a Receiver Operator Characteristic (ROC) curve of IgA ACCA (GNb4GNb), IgG ALCA (Gb3Gb) and IgG ASCA (Mannan) for differentiating between NIC and UC.
FIG. 5A is a graph showing the correlation between ASCA IgG and ALCA IgG in CD patients.
FIG. 5B is a graph showing the correlation between ASCA IgG and ACCA IgA in CD patients.
FIG. 5C is a graph showing the correlation between ALCA IgG and ACCA IgA in CD patients.
FIGS. 6A and 6B are histograms showing binding of anti-glycan antibodies to the indicated glycans in sera from patients with anti-phospholipid syndrome (APS).

DETAILED DESCRIPTION OF THE INVENTION

Crohn's disease (CD), irritable bowel syndrome (IBS) and anti-phospholipid syndrome (APS) are diagnosed by examining a test sample from a subject for antibodies to one or more specific glycans. The presence of the antibodies in the test sample indicates the subject has CD or APS. In some embodiments, elevated levels of glycans in a test sample from the subject as compared to the levels of the glycan or glycans in a reference sample that does not have CD indicates that the subject has CD. The methods can be used distinguish the presence of CD in a subject from other inflammatory bowel diseases (including ulcerative colitis).
A translation of the LinearCode™ syntax used to describe glycan structure to IUPAC nomenclature can be found in Table 1. The glycans are presented either in the International Union of Pure and Applied Chemistry (IUPAC) condensed form for nomenclature carbohydrate representation or in LINEARCODE® syntax, for linear code syntax principles see (Banin et al., Trends in Glycoscience and Glycotechnology, 14:127-37, 2002). Translation of LINEARCODE® to IUPAC representation is in Table 1. All the glycan structures that discussed in this disclosure, unless mentioned otherwise are connected to in the indicated anomericity α or β to other molecular structure, linker, or solid phase.
As used herein, the term “inflammatory bowel disease” is synonymous with “IBD” and is a collective term referring to both Crohn's disease and ulcerative colitis. Thus, an individual having either Crohn's disease or ulcerative colitis is defined herein as having IBD. Conversely, an individual having neither ulcerative colitis nor Crohn's disease does not have IBD as defined herein. The term “inflammatory bowel disease” distinguishes Crohn's disease and ulcerative colitis from all other disorders, syndromes or abnormalities of the gastroenterological tract including irritable bowel syndrome.
As used herein, the term “Non inflammatory bowel disease” is synonymous with “Non-IBD” and is a collective term referring to all other disorders, syndromes or abnormalities of the gastroenterological tract including irritable bowel syndrome (IBS).
The methods for diagnosing IBD may additionally include determining whether a sample is positive for anti-neutrophil cytoplasmic antibodies (ANCA). Anti-neutrophil cytoplasmic antibodies that produce a perinuclear staining pattern (pANCA) are elevated in 60-80% of UC patients and less frequently in CD and other disorders of the colon. Serum titers of ANCA are elevated in UC patients regardless of clinical status and, thus, do not reflect disease activity. High levels of serum ANCA also persist in UC patients five years post-colectomy. Although pANCA is found only very rarely in healthy adults and children, healthy relatives of UC patients have an increased frequency of pANCA, indicating that pANCA may be an immunogenetic susceptibility marker. ANCA reactivity is also present in a small portion of patients with Crohn's disease. The reported prevalence in CD varies, with most studies reporting that 10 to 30% of CD patients express ANCA (Saxon et al., J. Allergy Clin. Immunol. 86:202-210 (1990); Cambridge et al., Gut 33:668-674 (1992); Pool et al., Gut 3446-50 (1993); and Brokroelofs et al., Dig. Dis. Sci. 39:545-549 (1994)).
As used herein, the term “anti-neutrophil cytoplasmic antibody” is synonymous with “ANCA” and means antibodies to cytoplasmic components of a neutrophil. ANCA, such as serum or saliva ANCA, can be detected using an enzyme-linked immunosorbent assay with alcohol-fixed neutrophils. As disclosed herein, ANCA activity is divided into several broad categories: perinuclear to nuclear staining or cytoplasmic staining with perinuclear highlighting (pANCA); cytoplasmic neutrophil staining without perinuclear highlighting (cANCA); and diffuse staining with speckling across the entire neutrophil (SAPPA). The term ANCA, as used herein, encompasses all varieties of anti-neutrophils cytoplasmic reactivity, including pANCA, cANCA and SAPPA. Similarly, the term “ANCA” encompasses all immunoglobulin isotypes including, for example, immunoglobulin A and G.
The determination of whether a sample is positive for ANCA using non-histological means is made using antigen specific for ANCA using methods described in U.S. Pat. No. 6,218,129. Such an antigen specific for ANCA can be, for example, whole fixed neutrophils; an unpurified or partially purified neutrophil extract; a purified UC pANCA antigen such as a purified protein, protein fragment or synthetically produced peptide; an anti-ANCA idiotypic antibody; or the like. Particularly useful antigens specific for ANCA are peptides, which can be chemically synthesized or expressed on the surface of phage. Purified antigens specific for ANCA can be, for example, histone H1, or an ANCA-reactive fragment of histone H1, as described in U.S. Pat. No. 6,074,835 now U.S. Pat. No. 6,074,835; an ulcerative colitis pANCA secretory vesicle antigen or an ANCA-reactive fragment thereof; or a microbial UC pANCA antigen, such as a histone H1-like antigen, porin antigen, Bacteroides antigen, or ANCA-reactive fragment thereof, as described in U.S. Pat. No. 6,033,864 now U.S. Pat. No. 6,033,864. One skilled in the art understands that additional antigens specific for ANCA, including antigenic fragments and ANCA-reactive peptides, can be identified, for example, using a representative UC pANCA monoclonal antibody.
Generating an Anti-Glycan Antibody Profile
In performing the methods of the invention, a sample to be analyzed is obtained from the subject to be diagnosed. The term “sample,” as used herein, means any biological specimen obtained from an individual that contains antibodies. A sample can be, for example, whole blood, plasma, saliva or other bodily fluid or tissue having antibodies, preferably a serum sample. Samples can be diluted if desired before they are analyzed for anti-glycan antibodies. The subject can be, e.g., a human, a non-human primate (including a chimpanzee, ape, gorilla, old world primate), cow, horse, dog, cat, pig, goat, sheep, rodent (including, e.g., a mouse, rat, or guinea pig) Anti-glycan profiles can be determined by using methods known in the art for identifying antibodies to glycans. The methods include those disclosed in e.g., WO00/49412, or WO02/064556, or Schwarz et al., Glycobiology 13:749-54, 2003.
The methods are typically performed using reagents that specifically bind to the anti-glycan antibodies. The reagents can be, e.g., the specific glycan structures. Alternatively, the reagents can be other molecules or macromolecules that include the specific glycan structure. For example, the anti-Glc(β,1-3)Glc(β) antibody can be detected using the polysaccharide β-D(1-3)Glucan, a polymer of glucose units connected in a (β,1-3)Glycosidic bond. Thus, the glycan itself can be used for detecting the corresponding antibody or antibodies, as can any carbohydrate, peptide, protein, or any other molecular structure that includes the glycan.
If desired, the peptides that mimic carbohydrate antigens can be used in the methods and compositions described herein. The peptides can be used to identify specific anti glycan antibodies. Peptides which mimic structures recognized by antiglycan antibodies can be identified using methods known in the art, e.g., by screening a filamentous phage-displayed random peptide library (Zhan et al., Biochem Biophys Res Commun. 308:19-22, 2003; Hou et al., J. Immunol. 17:4373-79, 2003).
Glycan antigens used to identify various anti-glycan antibodies can be obtained from a variety of other sources so long as the antigen is capable of binding specifically to the given anti-glycan Binding to anti-glycan antibodies can be performed using variety of other immunoassay formats known in the art, including competitive and non-competitive immunoassay formats can also be used (Self and Cook, Curr. Opin. Biotechnol. 7:60-65 (1996), which is incorporated by reference). Other assays include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs). An enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or urease can be linked to a secondary antibody selective for a primary anti-glycan antibody of interest. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- a β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.). A useful secondary antibody linked to an enzyme can be obtained from a number of commercial sources; goat F(ab′)₂anti-human IgG-alkaline phosphatase, for example, can be purchased from Jackson Immuno-Research (West Grove, Pa.).
Immunoassays encompass capillary electrophoresis based immunoassays (CEIA) and can be automated, if desired. Immunoassays also can be used in conjunction with laser induced fluorescence (see, for example, Schmalzing and Nashabeh, Electrophoresis 18:2184-93 (1997)); Bao, J. Chromatogr. B. Biomed. Sci. 699:463-80 (1997), each of which is incorporated herein by reference). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, also can be used (Rongen et al., J. Immunol. Methods 204:105-133 (1997)).
A radioimmunoassay can also be used for determining whether a sample is positive for a glycan antibody, or for determining the level of anti-glycan antibodies in a sample. A radioimmunoassay using, for example, an ¹²⁵Iodine-labeled secondary antibody (Harlow and Lane, Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York, 1988, which is incorporated herein by reference) is encompassed within the invention.
A secondary antibody may alternatively be labeled with a chemiluminescent marker. Such a chemiluminescent secondary antibody is convenient for sensitive, non-radioactive detection of anti-glycan antibodies and can be obtained commercially from various sources such as Amersham Lifesciences, Inc. (Arlington Heights, Ill.).
A detectable reagent may also be labeled with a fluorochrome. Appropriate fluorochromes include, for example, DAPI, fluorescein, Hoechst. 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red or lissamine. A particularly useful fluorochrome is fluorescein or rhodamine. Secondary antibodies linked to fluorochromes can be obtained commercially. For example, goat F(ab′)₂anti-human IgG-FITC is available from Tago Immunologicals (Burlingame, Calif.).
A signal from the detectable reagent can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of ¹²⁵Iodine; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked reagents, a quantitative analysis of the amount of anti-glycan antibodies can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices, Menlo Park, Calif.) in accordance with the manufacturer's instructions. If desired, the assays of the invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
Other methods include, e.g., flow cytometry (including bead based immunoassays), and phage display technology for expressing a recombinant antigen specific for an anti-glycan antibody. Phage particles expressing the antigen specific for a desired anti-glycan antibody can be anchored, if desired, to a multiwell plate using an antibody such as an anti phage monoclonal antibody (Felici et al., “Phage-Displayed Peptides as Tools for Characterization of Human Sera” in Abelson (Ed.), Methods in Enzymol. 267, San Diego: Academic Press, Inc. (1996), which is incorporated by reference herein).
Anti-glycan antibodies are conveniently detected by simultaneously analyzing multiple sample for the presence of one or more anti-glycan antibodies. For example, the antibodies can be detected using an array of reagents that can bind specifically to the anti glycan antibodies. Preferably, each reagent is provided in a different location with a defined address on the array. By exposing the sample to array all the anti glycan antibodies that bind to the reagent on the array can be detected in one test Suitable arrays that include reagents (preferably carbohydrate reagents) that specifically detect the CD-detecting antibodies disclosed herein, e.g., an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Manna antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(p antibody, an anti-Gal 3-sulphate(β) antibody, an anti-aGlcNAc(β,1-3)GalNAc(p) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-Gal(α) antibody, an anti-Gal(β) antibody, an anti-GalNAc(α), an anti-Glc(α) antibody, an anti-Gal(β,1-6)Gal(β) antibody, an anti anti-GlcNAc(β,1-6)GalNAc(α) or an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β) antibody for diagnosing CD.
In some embodiments, the reagents that are used to specifically bind and detect those anti glycans antibodies are the specific glycan structures. In other embodiments, the reagents are other molecules or macromolecules that include the specific glycan structure. For example, the anti-Glc(β,1-3)Glc(β) antibody can be detected using the polysaccharide β-D(1-3)Glucan, a polymer of glucose units connected in a (β,1-3)Glycosidic bond. Thus, the glycan itself can be used for detecting the corresponding antibody or antibodies, as can any carbohydrate, peptide, protein, or any other molecular structure that includes the glycan.
The array may additionally include a reagent or reagent, e.g., a carbohydrate reagent or reagents, that detect an anti-Mannan antibodies or a ANCA. In some embodiments, the glycans are attached to the array via a linker. A suitable linker includes at least one ethylene glycol derivative, at least two cyanuric chloride derivatives and an anilino group.
Arrays useful for diagnosing APD can include a reagent (preferably a carbohydrate reagent) that specifically detects an anti-chitobiose antibody and, optionally, a reagent that specifically detects a β-2 glycoprotein for detecting.
If desired, peptides that mimic carbohydrate antigens can be used in the methods and compositions described herein. The peptides can be used to identify specific anti glycan antibodies. Peptides which mimic structures recognized by antiglycan antibodies can be identified using methods known in the art, e.g., by screening a filamentous phage-displayed random peptide library (Zhan et al., Biochem Biophys Res Commun. 308:19-22, 2003; Hou et al., J. Immunol. 17:4373-79, 2003.)
Interpreting Anti-Glycan Antibody Binding Data
Typically, binding of anti-glycan antibodies to glycans in a sample is compared to a reference population, and differences in levels of the anti-glycan antibodies in the two samples are compared. The threshold for determining whether a test sample is scored positive for CD or APS, or Non-IBD based on its ant-glycan antibody profile can be altered depending on the sensitivity or specificity desired. The clinical parameters of sensitivity, specificity, negative predictive value, positive predictive value and overall agreement are calculated using true positives, false positives, false negatives and true negatives. A “true positive” sample is a sample positive for CD according to colonoscopy, radiologic and/or histologic analysis, which is also diagnosed positive according to a method of the invention. A “false positive” sample is a sample negative for CD by colonoscopic, radiologic and/or histologic analysis, which is diagnosed positive according to a method of the invention. Similarly, a “false negative” is a sample positive for CD by colonoscopic, radiologic and/or histologic analysis, which is diagnosed negative according to a method of the invention. A “true negative” is a sample negative for CD by colonoscopic, radiologic and/or histologic analysis, and also negative for CD according to a method of the invention. See, for example, Mousy (Ed.), Intuitive Biostatistics New York: Oxford University Press (1995), which is incorporated herein by reference.
As used herein, the term “sensitivity” means the probability that a laboratory method is positive in the presence of CD. Sensitivity is calculated as the number of true positive results divided by the sum of the true positives and false negatives. Sensitivity essentially is a measure of how well a method correctly identifies those with disease. In a method of the invention, the anti-glycan antibody values can be selected such that the sensitivity of diagnosing an individual is at least about 60%, and can be, for example, at least about 65%, 70%, 75%, 80%, 85%, 90% or 95%.
As used herein, the term “specificity” means the probability that a method is negative in the absence of CD. Specificity is calculated as the number of true negative results divided by the sum of the true negatives and false positives. Specificity essentially is a measure of how well a method excludes those who do not have CD. The anti-glycan cut-off value can be selected such that, when the sensitivity is at least about 70%, the specificity of diagnosing an individual is in the range of 30-60%, for example, 35-60%, 40-60%, 45-60% or 50-60%.
The term “positive predictive value,” as used herein, is synonymous with “PPV” and means the probability that an individual diagnosed as having CD actually has the disease. Positive predictive value can be calculated as the number of true positives divided by the sum of the true positives and false positives. Positive predictive value is determined by the characteristics of the diagnostic method as well as the prevalence of the disease in the population analyzed. In a method of the invention, the anti-glycan antibody cut-off values can be selected such that the positive predictive value of the method in a population having a CD disease prevalence of 15% is at least about 5%, and can be, for example, at least about 8%, 10%, 15%, 20%, 25%,30% or 40%.
As used herein, the term “overall agreement” means the accuracy with which a method diagnoses a disease state. Overall agreement is calculated as the sum of the true positives and true negatives divided by the total number of sample results and is affected by the prevalence of CD in the population analyzed. The anti-glycan antibody cut-off values can be selected such that the overall agreement of a method of the invention in a patient population having an CD disease prevalence of 15% is at least about 45%, and can be, for example, at least about 50%, 55% or 60%.
The invention will be illustrated in the following non-limiting examples.

EXAMPLE 1

Comparative Antiglycan Antibody Levels in the Serum of Crohn's Disease Patients and Patients with Other Digestive Diseases

An anti-glycan antibody profile for IgG, IgA and IgM in the serum of the patients was obtained using GlycoChip® arrays (Glycominds, Ltd., Lod, Israel, Cat No. 9100). The arrays were constructed using procedures described in Schwarz et. al. Glycobiology, 13: 749-54, 2003. Anti-glycan antibody profiles of 45 CD patients and 27 patients with other digestive diseases were compared.
All serum samples were tested using GlycoChip® plates (Glycominds Ltd., Lod, Israel, Cat No. 9100), which was an array of mono and oligosaccharides covalently attached to a reduced volume 384-well micro titer plate. The mono and oligosaccharides displayed on the array are listed in Table 1. A translation of the LinearCode™ syntax used to describe glycan structure to IUPAC nomenclature can be found in Table 1.
The sera from patients volunteers who had signed an informed consent form were collected by Dr. Iris Dotan from the Gastroenterology and Liver Disease Institute in the Tel Aviv Sorasky Medical Center, Israel. All patients were diagnosed by Dr. Iris Dotan. The sera were collected in evacuated silicon coated gel containing tubes (Estar Technologies Cat# 616603GLV). The sera were separated from the blood cells and kept frozen at −25° C. until use. The volume of all solutions added to the glycan array was 10 μl/well. The sera were diluted (1:20; saturating concentration) in 0.15M Tris-HCl pH 7.2, 0.085M Mg2SO4, 0.05% Tween 20 (TBST) containing 1% BSA (Sigma), dispensed into glycan array plates using a Tecan Genesis Workstation 200 automated handling system, and incubated for 60 min at 37° C. The plates were then washed with 250 μL/well Phosphate buffered Saline with 0.05% Tween 20 (PBST, Sigma) in an automatic plate washer (Tecan, POWERWASHER™). At this point the following reagents, diluted in TBST with 1% BSA, were added using a Multidrop 384 dispenser (Thermo Labsystems) and incubated for 60 min at 37° C.: for IgG, IgA, and IgM determination—the respective sub-class specific biotinylated goat anti-human Ig antibody (Jackson, Pa., USA) at 2.8 μg/ml, 3 μg/ml, and 0.9 μg/ml, respectively. Following washing with PBST, Streptavidin-conjugated europium (0.1 μg/ml) diluted in TBST with 1% BSA was added to each well followed by incubation for 30 min at 37° C. in the dark, and washing with PBST. DELFIA™ enhancement solution was then added to the wells and the plates were incubated for 30 to 45 min in the dark at room temperature. The fluorescence of the wells was read with a Victor 1420 (Wallac, Finland) plate reader using time resolved fluorescence settings of 340/612 nm (Excitation/Emission).
Some patients were tested for the presence of antibodies to perinuclear anti neutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae (ASCA) IgG and IgA using a commercial kits made by INOVA, San-Diego, Calif. Cat. No 708290, 708865, 708870 respectively, according to the manufacturer instructions.
Tables 2, 3 and 4 present levels of IgG, IgA and IgM type antiglycan antibodies that were detected at significantly different levels between the CD population and the population with other digestive diseases. The values presented for IgG and IgA are absolute values. The values presented for IgM are absolute values after reduction of background. The back ground signal was measured as the signal received from wells with covalently bound p-nithrophenol. If the result was negative the signal was scored as zero.
Comparison of the average and median values of anti-carbohydrate antibodies in the CD and other digestive disease populations reveals a significant elevation in most of the anti glycans antibodies in the CD group as compared to the group containing individuals with the other digestive diseases group. None of the CD patients was found to be positive for pANCA antibodies. All the anti glycans levels that are displayed in Tables 2, 3 and 4 show statistically significant (α=0.05; p<0.05) differences between the CD groups and the other digestive disease or normal group. Statistically significant differences between the medians of signals of CD and other digestive disease population and normal population were observed for antibodies bound to the following glycans: Glc(β), Glc(β,1-4)Glc(β), Glc(β,1-3)Glc(β), GlcNAc(β) 6-sulfate, Man(α, 1-2)Man(α), Man(α,1-3)Man(α), Man(α,1-6)Man(α), Man(α), Man(α,1-3)[Man(α,1-6)]Man(α), Mannan, Dextran, Xylan, GlcNAc(β,1-4)GlcNAc(β), Gal 3-sulphate(P), GlcNAc(β,1-3)GalNAc(β), GlcNAc(β,1-3)Gal(β,1-4)Glc(β), Gal(α), Gal(β), GalNAc(α), Glc(α), Gal(β,1-6)Gal(β), GlcNAc(β,1-6)GalNAc(α) and Gal(α,1-3)Gal(β,1-4)GlcNAc(β).
Table 5 shows the specificity and sensitivity of the different IgG anti glycans for differentiation between CD and other digestive diseases using different cut-off values. The cutoff values for each glycans where set as the 89^thpercentile of the non CD group.
These results reveal a set of chemically defined glycan antigens that are useful for diagnosing CD. The levels of antibodies to those glycans are higher in the CD population than in the population of normal individuals or individuals with other digestive diseases. The antibodies that showed the greatest differentiation between CD and other digestive diseases in these studies are a set of antibodies to mannose based glycan fragment as well as antibodies to on Glc(β), Glc(β,1-4) Glc(β), Glc(β,1-3)Glc(β). Antibodies to Glc(β,1-3)Glc(β), Man(α,1-3)Man(α) and Man(α,1-3)[Man (a 1-6)]Man(α) were in particular able to differentiate between CD and other digestive disease at 57-62% sensitivity and 89%-93% specificity. The separation of those structures was better that what was achieved with Mannan (ASCA) 47% sensitivity and 89% specificity. Table 6 demonstrates that it is possible to use different cut of levels and to achieve higher sensitivity but lower specificity. Table 6 describe the sensitivity, specificity, True Positives (TP), True Negative (TN), False Positives (FP), and False Negatives (FN) and positive Predictive value (PPV) in different cut-of value for differentiation between CD and other digestive disease according to the level of Anti Glc(β,1-3)Glc (I), IgG and anti Mannan IgG. FIG. 1 is a Receiver Operator Characteristic (ROC) curve for differentiation between individuals with CD and individuals with other digestive diseases according to levels of anti Glc(β,1-3)Glc(β), IgG and anti Manna IgG antibodies.
By using combination of two or more glycans it is possible to improve the sensitivity with without reducing the specificity. For example, by setting cut-offs of 2000,000 for anti Glc(β,1-3)Glc(β) and 2,400,000 for anti Mannan and setting the criteria for identification of CD as those individuals who are above cut-off levels for either of the antibodies it is possible to achieve 82% sensitivity with 70% specificity. Achieving this sensitivity by each of the antibodies alone would require lower cut off points, but these lower cutoffs would lead to poor specificity (e.g., a specificity of 37% for Glc(β, I-3)Glc(β)).

EXAMPLE 2

Comparative Antiglycan Antibody Levels in the Serum of Crohn's Disease (CD) Colitis Patients and Ulcerative Colitis (UC) Patients

An anti-glycan antibody profile for IgG and IgA in the serum of the patients was obtained using GlycoChip® arrays (Glycominds, Ltd., Lod, Israel, Cat No. 9100). The arrays were constructed using procedures described in Schwarz et. al., Glycobiology 13: 749-54, 2003. Anti-glycan antibody profiles of 6 CD colitis patients and 19 UC patients were compared. All serum samples were collected and tested as described in Example 1.
Tables 7 and 8 show the levels of IgG and IgA type antiglycan antibodies that were detected at significantly different levels between the CD Colitis population and the UC population. The values presented for IgG and IgA are absolute values. Comparison of the average and median values of anti-carbohydrate antibodies in the CD Colitis patients and UC patients populations reveals a significant elevation in most of the anti glycans antibodies in the CD group as compared to the group containing individuals with the other digestive diseases group. All the anti glycans levels that are displayed in Tables 7 and 8 show statistically significant (α=0.05; p<0.05) differences between the CD Colitis group and the UC group, with the exception of anti Mannan (ASCA) IgA and IgG. The most significant difference between the antibodies levels in the IgG class was found in the levels of anti Man(α,1-3)Man(α), whereas for the IgA class the most significant difference was found between the levels of anti GlcNAc(β,1-4)GlcNAc(β) antibodies. No statistically significant difference between the levels of anti Mannan (IgG or IgA) levels of the CD Colitis patients and UC patients populations was detected in these studies. FIG. 2 is a box plot graph of the difference between CD colitis and UC groups for the levels of some antiglycan IgG and IgA antibodies.

EXAMPLE 3

Comparative Antiglycan Antibody Levels in the Serum of Crohn's Disease (CD) Patients, Ulcerative Colitis (UC) Patients, and Irritable Bowel Syndrome (IBS) Patients

The levels of antiglycan antibodies in serum from CD, UC, and IBS patients were compared.
An anti-glycan antibody profile for IgG, and IgA in the serum of the patients was obtained using GlycoChip® arrays (Glycominds, Ltd., Lod, Israel, Cat No. 9100). The arrays were constructed using procedures described in Schwarz et. al. Glycobiology 13:749-54, 2003. The levels of the following Anti-glycan antibody were measured: Anti Laminarobioside (Glc(β,1-3)Glc(β)) Carbohydrate Antibodies (ALCA); Anti Chitobioside (GlcNAc(β,1-4)GlcNAc(β)) Carbohydrate Antibodies (ACCA); and Anti mannan (Anti Saccromyces Cervicia Antigen (ASCA)). Those antibodies were measured in the serum 70 CD patients, 56 UC patients and 19 patients with Non-IBD digestive diseases Controls (NIC) were also compared. All serum samples were collected and tested using GlycoChip® plates (Glycominds Ltd., Lod, Israel, Cat No. 9100) as described in Example 1.
A summary of the results is presented in FIG. 3. CD patients have statistically significant higher levels of ASCA, ALCA and ACCA then UC patients. UC patients have statistically significant lower levels of ASCA, ALCA and ACCA than NIC patients. NIC patients have statistically significant higher IgA ACCA levels then UC patients.
The ROC curve in FIG. 4A demonstrates that all three markers can be used to differentiate between CD and UC. The ROC curve in FIG. 4B demonstrates that ACCA allows for differentiation between NIC and UC and shows the sensitivity and specificity for different cutoff levels. FIGS. 5A-C below show low or no correlation between the levels of IgG ASCA marker and IgG ALCA or IgA ACCA in CD patients. It is clear that each marker recognizes distinct sub-groups of Crohn's Disease patients.
Table 9 summarizes the sensitivity and specificity for each disease type using the combinations of the markers: a sample that tests ASCA(+) or ALCA(+) indicates the patient from whom the sample was obtained has CD and not UC. A patient who tests IgA ACCA(+) and ASCA(−) has a non-IBD digestive disease.
These data demonstrate that ASCA, ACCA, and ALCA markers can be used to differentially diagnose IBD (which includes CD and IBD) from all other intestinal diseases (NIC), including irritable bowel syndrome (IBS). The presence of antibodies to IgA ACCA and the absence of antibodies to ASCA indicates a subject has NIC. A subject is also diagnosed with NIC if ALCA antibodies are also absent from the sample.

EXAMPLE 4

Levels of Antiglycan Antibody Levels in the Serum of Anti-Phospholipid Syndrome (APS) Patients and Patients with Other Digestive Diseases

A pool of serum samples from APS patients was fractionated on a β-2 glycoprotein column and tested for the presence of anti-glycan antibodies.
Antibody binding was examined using GLYCOCHIP™ substrates as described in WO00/49412. Wells were blocked with ddH₂O/BSA 2.5%. The serum sample was diluted 1:2 in 1% TBST/BSA. Anti-IgA, IgG, and IgM samples were diluted 1:100 in TBST/BSA 1%. The Alexa 633 dye (Molecular Probes, Eugene, Oreg., # S-20992) was diluted 1:150 in TBST 1:150. Samples were injected using a Tescan HS4800 program. Dry arrays were scanned using an Affymetrix 428 Scanner, and images were analyzed using ‘ArrayPro’ software. Numerical values were exported to Excel and analyzed. For isotype determination, anti-human IgG, Fc gamma fragment specific\Biotin (Goat); Jackson; Cat # 109-065-008, anti-human IgM, Fc 5 mu fragment specific\Biotin (Goat); Jackson; Cat # 109-065-043, and anti-human serum IgA\biotin (Goat); Jackson; Cat # 109-065-011.
The serum sample was affinity-purified against a column of β-2-glycoprotein and then applied to a GlycoChip containing multiple glycans. The full magnitude (0-2.5×10⁷) of the interaction profile of the APS immunoglobulins and the tested glycans antibodies on the GlycoChip are shown in FIG. 6A. A smaller scale (0-5×10⁶) of the binding is shown in FIG. 6B.
The highest levels of antibodies were observed for antibodies against GlcNAc(β,1-4)GlcNAc(13) for each of the IgA, IgG, and IGM subclasses High IgG levels against LPS from Salmonella typhimurium, Man(α,1-6)Man(α), GlcNAc(α), GlcNAc(β) and Gal(β,1-4)Glc(β) (Lactose) were also observed.
IgA levels were highest against GlcNAc(β,1-4)GlcNAc(β) and relatively high against Gal(β,1-4)Glc(β) and LPS from Salmonella. IgM levels were highest against GlcNAc(β, I-4)GlcNAc(β) and relatively high against LPS from E. coli O26:B6, Glucose derivatives such as Glc(α), Glc(α,1-4)Glc(α), Glc(α,1-4)Glc(β), Glc(β,1-3)Glc(β) and Glc(β,1-4)Glc(β), GlcNAc(α), Rha(α) as well as Man(α,1-6)Man(α), GalA(β), GlcA(β) and LPS from Salmonella. Relatively low levels of Ig were detected against the preparation of Mannan used on the GlycoChip substrate.
These results demonstrate that elevated levels antibodies to GlcNAc(β,1-4)GlcNAc(β) in the blood may serve as a marker for diagnosis of APS, and/or for the severity of the disease.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

TABLE 1


Saccharides displayed on the glycan array

Glycan	IUPAC	LINEARCODE ®	Common Name

0	p-Nitronhenol	pNP-0
1	Gal(α)	Aa
2	Gal(β)	Ab
3	Gal(β, 1-3)GalNAc(α)	Ab3ANa
4	Gal(β, 1-3)GlcNAc(β)	Ab3GNb
5	Gal(β, 1-4)Glc(β)	Ab4Gb	Lactose
6	Gal(β, 1-6)Gal(β)	Ab6Ab
7	GalNAc(α)	ANa
8	GalNAc(β)	ANb
9	Fuc(α)	Fa
10	Fuc(β)	Fb
11	Glc(α)	Ga
12	Glc(α, 1-4)Glc(α)	Ga4Ga	Maltose
13	Glc(α, 1-4)Glc(β)	Ga4Gb
14	Glc(β)	Gb
15	Glc(β, 1-4)Glc(β)	Gb4Gb	Cellobiose
16	Glc(β, 1-4)Glc(β, 1-4)Glc(β)	Gb4Gb4Gb	Cellotriose
17	GlcNAc(α)	GNa
18	GlcNAc(β)	GNb
19	GlcNAc(β, 1-3)GalNAc(α)	GNb3ANa
20	GlcNAc(β, 1-4)GlcNAc(β)	GNb4GNb	Chitobiose
21	L-Rha(α)	Ha
22	GalA(β)	Lb
23	Man(α)	Ma
24	Man(β)	Mb
25	Neu5Ac(α)	NNa
26	L-Araf(α)	Ra
27	GlcA(β)	Ub
28	X(α)	Xa
29	X(β)	Xb
30	Gal(β, 1-3)[GlcNAc(β, 1-6)]GalNAc(α)	Ab3(GNb6)ANa
31	Gal(β, 1-4)GlcNAc(α)	Ab4GNa
32	Gal(α, 1-3)Gal(β, 1-4)GlcNAc(β)	Aa3Ab4GNb	Linear B-2
33	Gal(β, 1-3)Gal(β, 1-4)GalNAc(β)	Ab4GNb
34	Man(β, 1-4)GlcNAc(β)	Mb4Gb
35	GlcNAc(β, 1-6)GalNAc(α)	GNb6ANa
36	Fuc(α, 1-2)Gal(β)	Fa2Ab
37	Man(α, 1-3)Man(α)	Ma3Ma
38	GlcNAc(β) 6-sulfate	GN[6S]b
39	Glc(β, 1-3)Glc(β)	Gb3Gb
40	Gal(β) 3-sulfate	A[3S]b
41	Man(α, 1-3)[Man(α, 1-6)]Man(β)	Ma3(Ma6)Mb
42	GlcNAc(β, 1-3)Gal(α, 1-4)Glc(β)	GNb3Ab4Gb	Lacto-3
43	Gal(α, 1-4)Gal(β, 1-4)Glc(β)	Aa4Ab4Gb	Pk antigen
44	Man(α, 1-6)Man α	Ma6Ma
45	Man(α, 1-2)Man α	Ma2Ma
46	Dextran		Dextran
47	Mannam		Mannam
48	Xylan		Xylan

TABLE 2


Fluorescent signals from binding of IgG antibodies to different glycans
in CD patients and non CD patients. Glycans are presented in LINEARCODE ®.

Patient No.	Clinical condition	IgG Gb	IgG Gb3Gb	IgG Gb4Gb	IgG GN[6S]b	IgG Ma

10,001	Crohn's disease	1,360,254	1,977,964	1,336,648	1,027,193	1,410,642
10,004	Crohn's disease	3,663,580	5,704,570	5,099,956	2,099,879	2,164,970
10,005	Crohn's disease	1,526,204	1,871,425	1,454,552	1,697,361	1,335,277
10,006	Crohn's disease	774,615	281,544	488,393	297,446	261,425
10,007	Crohn's disease	1,183,143	1,474,083	1,010,984	1,337,825	1,522,187
10,008	Crohn's disease	1,047,535	3,935,252	1,173,449	656,185	610,749
10,009	Crohn's disease	1,120,092	1,061,053	913,603	773,633	434,718
10,011	Crohn's disease	1,555,366	3,915,250	1,263,015	1,514,790	1,319,000
10,012	Crohn's disease	738,615	997,829	828,833	710,883	815,777
10,013	Crohn's disease	491,219	494,196	487,233	405,409	550,041
10,015	Crohn's disease	2,507,309	3,287,329	3,150,129	1,752,931	1,210,172
10,016	Crohn's disease	1,695,721	2,983,993	2,389,536	1,363,739	1,073,805
10,018	Crohn's disease	2,349,612	1,749,025	1,975,697	1,316,616	1,642,423
10,021	Crohn's disease	936,577	2,010,561	1,236,079	736,454	647,085
10,025	Crohn's disease	1,125,348	7,400,065	1,364,422	873,065	696,782
10,026	Crohn's disease	584,311	2,397,273	1,007,841	570,786	396,704
10,027	Crohn's disease	331,704	3,884,268	1,013,711	391,803	323,622
10,028	Crohn's disease	527,070	2,540,157	776,682	842,969	471,600
10,031	Crohn's disease	1,160,637	1,403,953	1,163,714	903,043	1,090,200
10,033	Crohn's disease	1,658,517	1,171,191	795,746	1,166,575	845,487
10,034	Crohn's disease	487,703	454,402	1,030,665	343,963	377,012
10,036	Crohn's disease	1,669,205	2,969,747	1,661,164	1,729,819	2,023,690
10,041	Crohn's disease	2,099,634	8,111,207	1,506,694	1,366,204	2,194,579
10,042	Crohn's disease	3,414,756	6,232,427	4,724,789	3,236,890	2,752,951
10,043	Crohn's disease	2,375,664	8,432,547	5,565,720	1,826,113	2,179,476
10,047	Crohn's disease	936,002	6,541,240	736,485	1,344,797	1,007,696
10,058	Crohn's disease	1,229,222	3,933,918	1,268,315	1,097,145	1,122,154
10,060	Crohn's disease	3,776,848	7,519,448	3,738,773	3,273,536	2,478,007
10,061	Crohn's disease	377,655	7,004,834	426,579	359,296	413,067
10,062	Crohn's disease	2,157,588	3,580,257	2,335,797	2,291,757	2,178,945
10,064	Crohn's disease	1,201,946	2,571,544	2,116,543	1,307,836	2,149,447
10,067	Crohn's disease	1,662,361	4,387,868	1,723,425	1,379,202	1,863,573
10,068	Crohn's disease	646,782	805,864	612,996	820,938	1,066,384
10,071	Crohn's disease	1,759,894	4,847,180	1,435,670	1,215,684	1,677,667
10,073	Crohn's disease	842,124	3,215,453	1,223,370	957,636	1,823,465
10,074	Crohn's disease	1,490,333	2,253,024	627,434	697,763	1,567,676
10,075	Crohn's disease	5,537,343	9,220,529	4,909,931	2,091,679	3,060,118
10,077	Crohn's disease	804,027	1,800,557	827,615	1,099,475	844,037
10,078	Crohn's disease	1,129,868	3,107,666	2,487,836	1,523,754	1,578,420
10,081	Crohn's disease	1,416,365	2,436,837	1,243,841	933,009	1,418,666
10,089	Crohn's disease	3,140,987	2,809,714	3,881,344	1,459,563	1,732,146
10,090	Crohn's disease	1,142,664	6,465,008	6,126,851	1,040,285	1,733,530
10,094	Crohn's disease	2,581,852	3,002,260	2,533,016	1,828,766	2,171,545
10,095	Crohn's disease	4,307,357	5,194,520	2,939,889	1,440,026	1,664,132
10,102	Crohn's disease	602,214	902,531	797,794	684,982	768,939
10,051	No digestive disea	2,345,273	2,322,503	1,994,931	2,743,406	1,186,818
10,052	No digestive disea	941,167	697,884	747,644	414,298	385,995
10,053	No digestive disea	346,709	531,417	276,989	339,655	283,955
10,054	No digestive disea	692,918	664,121	993,676	680,576	539,916
10,059	Anal fissure	931,210	1,033,766	686,670	585,045	482,405
10,066	Proctitis/Psoriasis	977,625	955,662	1,003,683	880,485	698,411
10,080	No digestive disea	1,742,919	2,061,316	1,679,459	862,024	678,925
10,082	No digestive disea	606,761	2,058,347	951,804	631,202	681,631
10,003	Ulcerative colitis	906,251	695,019	504,690	729,287	432,922
10,020	Ulcerative colitis	1,354,222	4,073,378	1,231,701	1,258,840	1,363,896
10,022	Ulcerative colitis	971,547	2,471,052	1,617,809	805,585	696,492
10,023	Ulcerative colitis	476,805	1,684,016	407,103	335,428	741,738
10,024	Ulcerative colitis	1,536,705	1,866,888	1,294,792	1,278,711	981,713
10,030	Ulcerative colitis	313,802	319,115	370,440	288,801	383,159
10,039	Ulcerative colitis	788,940	1,322,675	2,049,445	579,957	427,353
10,040	Ulcerative colitis	508,502	878,458	506,749	390,383	397,998
10,044	Ulcerative colitis	1,134,152	1,000,922	1,430,170	794,829	1,166,622
10,050	Ulcerative colitis	1,307,947	1,067,601	1,111,619	912,013	1,056,915
10,065	Ulcerative colitis	983,243	1,390,482	1,028,795	842,732	666,535
10,069	Ulcerative colitis	598,736	708,048	978,417	672,092	359,555
10,072	Ulcerative colitis	320,461	473,172	342,039	363,893	455,588
10,079	Ulcerative colitis	405,166	3,763,266	904,868	613,527	425,264
10,084	Ulcerative colitis	703,594	1,982,678	2,259,105	545,266	592,993
10,086	Ulcerative colitis	686,425	808,037	713,774	466,633	402,319
10,087	Ulcerative colitis	615,110	428,332	577.232	386,386	409,432
10,096	Ulcerative colitis	997,504	2,041,057	949,569	673,275	633,246
10,097	Ulcerative colitis	424,300	1,024,501	805,975	458,307	267,256
	Avarage
	Crohn's disease	1,625,556	3,518,257	1,898,152	1,239,749	1,345,981
	No Chrohn's Disea	874,703	1,419,404	1,022,931	719,712	625,491
	Median
	Crohn's disease	1,229,222	2,983,993	1,268,315	1,166,575	1,335,277
	No Chrohn's Disea	788,940	1,033,766	951,804	631,202	539,916
	ttest CD vs Non C	0.001630097	4.0972E−05	0.004132072	0.000688569	3.87726E−06

Patient No.	Clinical condition	IgG Ma3(Ma6)Mb	IgG Ma3Ma	IgG Mannan	IgG Xylan	IgG Ma2Ma

10,001	Crohn's disease	1,144,296	1,583,677	7,628,072	5,577,215	849,550
10,004	Crohn's disease	1,465,706	3,207,783	7,476,981	4,596,345	3,150,105
10,005	Crohn's disease	921,447	991,861	5,827,429	4,982,760	857,537
10,006	Crohn's disease	153,551	267,094	1,310,353	543,373	495,362
10,007	Crohn's disease	957,160	1,102,706	3,891,170	2,264,442	894,460
10,008	Crohn's disease	487,133	508,943	7,900,531	2,858,992	918,954
10,009	Crohn's disease	653,203	735,213	3,625,333	860,059	1,786,497
10,011	Crohn's disease	600,433	695,166	6,044,060	2,016,766	795,378
10,012	Crohn's disease	512,116	449,986	1,016,305	509,887	874,119
10,013	Crohn's disease	369,100	360,682	975,473	858,010	497,853
10,015	Crohn's disease	1,280,894	1,706,919	8,298,001	6,144,885	1,865,891
10,016	Crohn's disease	1,019,572	933,689	8,178,923	3,474,026	1,423,401
10,018	Crohn's disease	857,701	1,424,193	2,438,472	1,837,913	2,114,539
10,021	Crohn's disease	680,544	668,362	4,677,824	1,836,902	688,760
10,025	Crohn's disease	618,026	961,392	7,750,817	610,583	1,306,723
10,026	Crohn's disease	366,620	500,604	4,427,669	409,875	754,569
10,027	Crohn's disease	409,228	549,358	4,184,500	1,477,393	409,405
10,028	Crohn's disease	371,962	644,191	1,133,383	580,308	388,120
10,031	Crohn's disease	922,033	1,360,531	6,070,889	5,267,773	1,024,636
10,033	Crohn's disease	628,923	860,940	8,668,310	1,372,493	999,469
10,034	Crohn's disease	317,412	402,687	4,878,747	873,284	317,845
10,036	Crohn's disease	1,699,947	1,824,104	7,240,661	3,315,784	1,815,367
10,041	Crohn's disease	1,943,357	1,877,528	6,166,731	1,716,244	1,866,462
10,042	Crohn's disease	2,886,293	3,805,675	4,968,270	2,207,087	1,975,431
10,043	Crohn's disease	2,694,502	4,144,108	7,766,334	1,594,568	6,234,204
10,047	Crohn's disease	870,673	1,011,207	1,929,720	510,038	1,276,888
10,058	Crohn's disease	1,202,918	1,158,350	1,060,988	3,370,770	1,097,275
10,060	Crohn's disease	3,298,731	2,075,262	6,622,444	2,830,021	2,587,250
10,061	Crohn's disease	380,758	309,119	1,440,898	277,672	1,125,410
10,062	Crohn's disease	1,790,623	1,986,588	5,928,994	4,135,199	1,725,643
10,064	Crohn's disease	3,229,183	2,010,487	7,015,353	4,157,443	1,546,371
10,067	Crohn's disease	2,466,987	2,121,119	4,103,563	1,406,894	3,016,091
10,068	Crohn's disease	2,089,715	1,525,952	856,926	520,606	1,364,558
10,071	Crohn's disease	1,557,477	2,457,616	5,460,883	3,745,128	1,980,052
10,073	Crohn's disease	2,274,968	1,845,219	7,159,301	6,675,699	2,240,331
10,074	Crohn's disease	1,854,560	1,499,634	5,758,566	998,526	1,155,521
10,075	Crohn's disease	2,760,344	4,585,798	7,712,384	6,780,460	4,835,416
10,077	Crohn's disease	632,785	1,010,415	1,238,077	908,558	726,513
10,078	Crohn's disease	1,459,959	1,509,299	7,284,452	2,493,069	1,974,718
10,081	Crohn's disease	692,890	1,391,276	6,617,906	4,927,776	1,038,228
10,089	Crohn's disease	812,187	1,751,178	1,948,648	1,671,266	1,975,468
10,090	Crohn's disease	947,788	1,075,332	7,029,051	1,226,773	986,138
10,094	Crohn's disease	1,864,642	1,426,014	3,755,208	1,359,423	1,889,334
10,095	Crohn's disease	1,246,967	1,260,260	5,473,997	1,284,127	1,587,782
10,102	Crohn's disease	650,448	612,321	779,631	515,705	999,742
10,051	No digestive disea	1,041,917	1,987,733	3,590,967	1,737,022	1,601,098
10,052	No digestive disea	300,723	438,891	736,762	776,440	345,938
10,053	No digestive disea	272,260	326,337	792,256	625,138	314,716
10,054	No digestive disea	425,475	213,664	1,280,390	1,600,067	484,686
10,059	Anal fissure	434,125	288,509	2,858,138	725,949	411,652
10,066	Proctitis/Psoriasis	775,253	855,457	1,721,699	1,307,196	878,148
10,080	No digestive disea	529,711	671,004	1,953,921	1,987,675	556,509
10,082	No digestive disea	500,014	512,486	4,193,860	1,440,782	616,439
10,003	Ulcerative colitis	336,989	349,315	1,022,364	504,020	640,303
10,020	Ulcerative colitis	902,559	705,623	2,556,960	1,911,575	641,831
10,022	Ulcerative colitis	656,384	356,351	5,619,844	2,466,668	689,170
10,023	Ulcerative colitis	185,491	265,536	708,233	482,414	524,736
10,024	Ulcerative colitis	422,316	709,227	6,380,359	2,793,937	1,501,433
10,030	Ulcerative colitis	267,131	302,649	1,539,392	918,717	465,098
10,039	Ulcerative colitis	484,029	500,544	1,614,749	627,274	962,879
10,040	Ulcerative colitis	417,665	292,485	2,881,788	977,455	1,617,762
10,044	Ulcerative colitis	749,019	912,335	1,385,640	1,761,062	830,609
10,050	Ulcerative colitis	756,151	951,103	1,829,813	972,519	1,200,920
10,065	Ulcerative colitis	672,182	852,588	2,333,880	1,363,312	1,165,936
10,069	Ulcerative colitis	339,385	366,514	1,736,635	1,231,461	599,938
10,072	Ulcerative colitis	392,522	506,615	3,309,525	471,592	482,991
10,079	Ulcerative colitis	370,064	356,485	1,665,869	576,365	293,343
10,084	Ulcerative colitis	431,487	430,193	1,655,824	548,041	509,652
10,086	Ulcerative colitis	379,369	356,194	2,174,9298	1,547,265	476,815
10,087	Ulcerative colitis	329,779	512,020	1,319,329	382,061	380,169
10,096	Ulcerative colitis	502,614	613,755	5,289,045	757,079	685,412
10,097	Ulcerative colitis	386,277	295,685	1,478,519	510,925	422,520
	Avarage
	Crohn's disease	1,243,023	1,426,441	4,904,849	2,390,714	1,542,963
	No Chrohn's Disea	487,551	552,944	2,356,692	1,148,297	714,840
	Median
	Crohn's disease	947,788	1,260,260	5,473,997	1,716,244	1,276,888
	No Chrohn's Disea	425,475	438,691	1,736,635	972,519	599,938
	ttest CD vs Non C	2.11271E−05	3.23203E−05	1.02024E−05	0.00132903	0.000372938

						IgG
						ASCA
						(IU)
Patient No.	Clinical condition	IgG Aa	IgG Ab	IgG ANa	IgG Ga	pANC

10,001	Crohn's disease	1,026,505	820,014	466,637	1,258,316
10,004	Crohn's disease	1,853,626	1,851,915	1,134,167	2,304,370
10,005	Crohn's disease	1,060,156	1,075,287	1,097,821	1,936,815
10,006	Crohn's disease	161,595	114,468	135,241	302,565
10,007	Crohn's disease	1,206,075	1,032,927	804,835	1,442,191
10,008	Crohn's disease	1,143,656	1,918,663	330,682	2,831,482	80 neg
10,009	Crohn's disease	809,917	1,065,462	389,609	834,189
10,011	Crohn's disease	1,235,678	1,111,199	648,037	2,843,354
10,012	Crohn's disease	561,052	700,662	589,469	1,559,067
10,013	Crohn's disease	258,936	269,477	390,232	443,436
10,015	Crohn's disease	2,313,400	1,657,409	1,472,395	1,738,460
10,016	Crohn's disease	4,167,686	1,150,916	837,155	1,754,605
10,018	Crohn's disease	1,282,598	1,116,190	1,304,881	1,556,960
10,021	Crohn's disease	431,224	417,858	624,273	1,097,281
10,025	Crohn's disease	472,016	407,187	666,244	1,052,914	49 neg
10,026	Crohn's disease	814,529	575,940	804,015	794,465
10,027	Crohn's disease	1,011,807	325,270	225,780	860,654
10,028	Crohn's disease	400,412	374,748	565,621	933,864
10,031	Crohn's disease	614,602	645,633	434,719	1,029,243
10,033	Crohn's disease	487,495	699,125	710,940	1,311,185	108 neg
10,034	Crohn's disease	130,726	240,504	254,960	353,496
10,036	Crohn's disease	1,312,161	847,504	1,148,197	2,764,778
10,041	Crohn's disease	1,002,663	944,926	614,258	1,790,996
10,042	Crohn's disease	2,453,713	1,169,199	1,491,661	2,608,430
10,043	Crohn's disease	1,533,054	850,045	857,270	4,436,841
10,047	Crohn's disease	609,950	611,669	498,004	1,408,015
10,058	Crohn's disease	739,330	352,829	525,407	1,349,540	9 neg
10,060	Crohn's disease	2,051,391	1,446,321	1,320,361	3,268,250
10,061	Crohn's disease	264,741	171,160	196,852	711,761
10,062	Crohn's disease	1,300,507	939,781	1,000,803	1,809,051
10,064	Crohn's disease	3,778,662	695,314	536,893	1,212,355
10,067	Crohn's disease	1,374,889	342,454	509,186	2,489,775
10,068	Crohn's disease	236,510	358,229	488,415	1,037,834
10,071	Crohn's disease	1,109,196	1,555,029	698,647	1,691,995
10,073	Crohn's disease	421,833	391,254	494,614	1,551,800	72 neg
10,074	Crohn's disease	385,865	620,552	291,814	637,642	43 neg
10,075	Crohn's disease	1,560,441	1,816,357	994,508	2,812,624
10,077	Crohn's disease	1,221,596	352,019	371,924	1,047,431	15 neg
10,078	Crohn's disease	485,474	430,568	567,379	1,318,049	89 neg
10,081	Crohn's disease	799,172	688,082	440,606	759,311	39 neg
10,089	Crohn's disease	6,235,212	1,056,023	879,862	1,551,638	12 neg
10,090	Crohn's disease	616,609	527,011	612,911	1,024,901
10,094	Crohn's disease	603,455	449,558	730,609	1,321,795
10,095	Crohn's disease	704,930	541,730	441,276	2,730,801
10,102	Crohn's disease	425,568	257,965	402,233	1,119,216
10,051	No digestive disea	325,091	277,928	291,398	765,670
10,052	No digestive disea	791,714	924,915	549,282	3,092,123
10,053	No digestive disea	226,043	634,923	193,641	1,110,574
10,054	No digestive disea	194,929	147,713	136,850	665,259
10,059	Anal fissure	491,013	482,992	344,004	796,813
10,066	Proctitis/Psoriasis	532,501	611,229	555,691	889,848
10,080	No digestive disea	544,141	349,525	358,554	729,190
10,082	No digestive disea	736,553	722,306	386,966	1,006,755
10,003	Ulcerative colitis	187,886	165,987	257,702	659,133
10,020	Ulcerative colitis	1,107,251	1,087,519	758,804	1,269,501
10,022	Ulcerative colitis	612,362	645,057	377,726	1,467,537	40 neg
10,023	Ulcerative colitis	230,640	167,922	159,075	693,800
10,024	Ulcerative colitis	984,301	963,564	803,682	1,278,165
10,030	Ulcerative colitis	218,405	486,030	131,947	660,381
10,039	Ulcerative colitis	535,526	415,798	335,651	694,135
10,040	Ulcerative colitis	364,266	487,974	236,382	487,736
10,044	Ulcerative colitis	702,427	721,510	678,997	1,026,423
10,050	Ulcerative colitis	416,618	518,265	637,011	992,490
10,065	Ulcerative colitis	433,662	723,424	593,461	953,378
10,069	Ulcerative colitis	205,729	474,181	329,163	1,215,250	8 neg
10,072	Ulcerative colitis	192,196	198,520	231,397	1,015,614
10,079	Ulcerative colitis	150,330	173,270	460,674	983,715
10,084	Ulcerative colitis	438,643	376,558	448,064	735,412
10,086	Ulcerative colitis	203,077	107,695	323,692	877,230
10,087	Ulcerative colitis	254,434	239,993	338,760	578,501	6 neg
10,096	Ulcerative colitis	384,127	475,422	459,440	1,205,875
10,097	Ulcerative colitis	174,679	275,494	344,389	751,112
	Avarage
	Crohn's disease	1,168,009	775,738	666,031	1,570,527
	No Chrohn's Disea	431,083	476,137	397,133	985,319
	Median
	Crohn's disease	814,529	688,082	589,469	1,349,540
	No Chrohn's Disea	384,127	475,422	344,389	889,848
	ttest CD vs Non C	0.00157261	0.00367082	0.000291031	0.001831333

TABLE 3


Fluorescent signals from binding of IgA and IgM antibodies to different glycans
in CD patients and non CD patients. Glycans are presented in LINEARCODE ®.

Patient No.	Clinical condition	IgA ASCA (IU)	IgA GNb3ANa	IgA GNb4GNb	IgA Ma5Ma	IgA Mannan

10,001	Crohn's disease		234,038	217,568	282,269	2,901,044
10,004	Crohn's disease		758,739	769,776	665,102	8,412,607
10,005	Crohn's disease		380,334	743,508	591,533	1,821,783
10,006	Crohn's disease		255,036	243,466	308,555	1,177,812
10,007	Crohn's disease		266,542	363,415	367,037	1,361,741
10,008	Crohn's disease	33	674,715	769,785	779,172	2,027,609
10,009	Crohn's disease		649,639	641,504	669,799	1,044,123
10,011	Crohn's disease		533,187	774,440	814,480	3,353,356
10,012	Crohn's disease		448,590	617,530	434,496	927,252
10,013	Crohn's disease		1,120,824	1,219,712	1,119,489	1,197,765
10,015	Crohn's disease		973,774	1,365,559	988,046	1,685,774
10,016	Crohn's disease		993,380	832,750	816,158	3,390,170
10,018	Crohn's disease		872,212	826,559	884,411	1,355,268
10,021	Crohn's disease		332,912	318,413	283,699	652,771
10,025	Crohn's disease	54	798,846	525,004	575,726	1,857,147
10,026	Crohn's disease		752,315	660,677	693,119	887,797
10,027	Crohn's disease		289,687	220,786	273,054	912,829
10,028	Crohn's disease		153,168	355,502	107,442	861,641
10,031	Crohn's disease		314,983	339,395	450,527	1,322,991
10,033	Crohn's disease	43	549,648	505,120	641,307	3,306,701
10,034	Crohn's disease		245,321	299,225	183,931	1,010,743
10,036	Crohn's disease		722,490	1,092,178	888,276	1,469,569
10,041	Crohn's disease		342,492	461,173	317,150	1,410,747
10,042	Crohn's disease		618,649	804,299	660,287	1,012,221
10,043	Crohn's disease		1,052,988	1,108,655	790,738	2,747,565
10,047	Crohn's disease		610,743	304,354	747,408	3,723,514
10,058	Crohn's disease	18	440,772	433,871	459,197	1,080,842
10,060	Crohn's disease		979,471	656,668	2,481,088	5,456,752
10,061	Crohn's disease		1,409,783	1,785,954	1,864,605	3,090,000
10,062	Crohn's disease		973,871	1,265,139	1,048,774	7,127,363
10,064	Crohn's disease		727,884	1,126,501	745,449	7,702,531
10,067	Crohn's disease		241,286	289,944	334,231	1,120,597
10,068	Crohn's disease		860,717	920,038	812,376	1,306,235
10,071	Crohn's disease		1,047,039	798,301	995,826	2,852,441
10,073	Crohn's disease	113	1,181,172	1,345,794	844,175	5,292,942
10,074	Crohn's disease	18	620,845	648,591	563,327	3,405,349
10,075	Crohn's disease		264,428	296,290	295,247	3,304,817
10,077	Crohn's disease	5	440,662	316,162	186,000	647,541
10,078	Crohn's disease	104	704,473	636,347	1,356,702	2,979,296
10,081	Crohn's disease	17	509,535	360,565	280,050	1,558,484
10,089	Crohn's disease	29	1,030,030	1,025,812	692,547	930,450
10,090	Crohn's disease		406,004	376,014	275,862	2,584,137
10,094	Crohn's disease		881,860	584,765	435,724	826,038
10,095	Crohn's disease		38,087	41,257	14,441	540,612
10,102	Crohn's disease		482,397	457,212	428,631	899,291
10,051	No digestive disease		774,286	1,543,419	674,603	1,290,323
10,052	No digestive disease		393,022	425,926	368,886	881,150
10,053	No digestive disease		878,508	1,064,617	748,198	1,055,576
10,054	No digestive disease		441,376	439,396	439,527	1,086,886
10,059	Anal fissure		293,422	265,604	410,485	1,062,125
10,066	Proctitis/Psoriasis		986,215	915,090	363,521	1,950,182
10,080	No digestive disease		707,800	741,718	472,696	1,882,808
10,082	No digestive disease		997,253	194,653	179,355	994,997
10,003	Ulcerative colitis		150,201	168,212	117,909	941,586
10,020	Ulcerative colitis		141,658	182,637	483,747	1,227,006
10,022	Ulcerative colitis	18	206,741	242,903	361,417	1,276,258
10,023	Ulcerative colitis		533,213	250,089	229,484	963,955
10,024	Ulcerative colitis		338,631	355,952	420,148	1,035,057
10,030	Ulcerative colitis		206,757	302,015	432,372	1,372,125
10,039	Ulcerative colitis		809,397	475,750	340,776	1,239,784
10,040	Ulcerative colitis		169,128	226,449	177,048	1,052,267
10,044	Ulcerative colitis		333,466	446,973	337,007	731,340
10,050	Ulcerative colitis		293,205	249,396	280,097	665,569
10,065	Ulcerative colitis		297,672	356,560	285,878	777,227
10,069	Ulcerative colitis	8	248,549	230,391	330,608	711,540
10,072	Ulcerative colitis		263,715	210,346	256,584	1,240,896
10,079	Ulcerative colitis		366,346	516,296	425,728	993,085
10,084	Ulcerative colitis		203,365	235,662	248,564	702,479
10,086	Ulcerative colitis		563,803	342,868	106,336	722,441
10,087	Ulcerative colitis	19	517,514	190,637	266,757	1,128,041
10,096	Ulcerative colitis		536,802	460,213	594,745	910,216
10,097	Ulcerative colitis		305,982	324,637	379,409	910,626
	Avarage
	Crohn's disease		626,280	661,007	654,844	2,320,850
	No Chrohn's Disease		442,890	421,422	360,447	1,067,616
	Median
	Crohn's disease		618,649	636,347	641,307	1,469,569
	No Chrohn's Disease		338,631	324,637	361,417	1,035,057
	ttest CD vs Non CD		0.014066493	0.006955663	0.00158187	0.001111753

Patient No.	Clinical condition	IgA Ab	IgA Ab6Ab	IgA GNb6ANa	IgA Aa3Ab4GNb3Ab4Gb

10,001	Crohn's disease	162,963	222,014	457,637	160,809
10,004	Crohn's disease	336,469	723,124	704,572	312,718
10,005	Crohn's disease	376,168	376,729	481,419	479,158
10,006	Crohn's disease	181,747	301,883	397,318	147,591
10,007	Crohn's disease	145,443	267,124	456,536	175,615
10,008	Crohn's disease	357,896	656,873	690,791	405,779
10,009	Crohn's disease	531,803	459,344	760,867	505,990
10,011	Crohn's disease	399,677	384,850	732,815	451,006
10,012	Crohn's disease	793,281	351,295	733,115	258,768
10,013	Crohn's disease	666,523	921,613	1,574,544	755,187
10,015	Crohn's disease	671,617	644,176	1,884,674	823,921
10,016	Crohn's disease	788,283	953,482	960,341	1,035,719
10,018	Crohn's disease	905,785	944,644	1,114,615	1,120,640
10,021	Crohn's disease	253,534	238,864	367,215	291,684
10,025	Crohn's disease	365,034	421,319	614,665	422,498
10,026	Crohn's disease	341,464	350,620	1,150,207	365,731
10,027	Crohn's disease	153,556	230,660	349,796	162,963
10,028	Crohn's disease	54,145	99,647	194,085	70,783
10,031	Crohn's disease	160,146	222,400	447,640	165,475
10,033	Crohn's disease	273,900	429,171	778,721	370,672
10,034	Crohn's disease	171,098	122,238	369,875	243,404
10,036	Crohn's disease	374,533	610,588	643,454	439,899
10,041	Crohn's disease	199,838	291,742	205,720	181,180
10,042	Crohn's disease	420,518	458,300	1,391,126	529,478
10,043	Crohn's disease	605,571	542,596	569,482	628,435
10,047	Crohn's disease	392,383	327,753	458,138	446,556
10,058	Crohn's disease	319,531	471,263	326,815	334,876
10,060	Crohn's disease	644,454	689,063	709,019	664,299
10,061	Crohn's disease	1,206,122	1,066,288	1,343,921	1,252,570
10,062	Crohn's disease	1,022,422	795,474	886,127	648,731
10,064	Crohn's disease	276,204	387,207	429,791	361,165
10,067	Crohn's disease	212,729	275,459	299,711	183,918
10,068	Crohn's disease	323,221	314,013	563,963	485,132
10,071	Crohn's disease	641,204	545,489	738,740	619,438
10,073	Crohn's disease	947,978	502,913	549,025	1,734,370
10,074	Crohn's disease	457,812	527,719	487,532	484,359
10,075	Crohn's disease	194,980	259,283	247,300	225,781
10,077	Crohn's disease	157,701	285,013	251,925	125,116
10,078	Crohn's disease	303,071	458,245	551,350	354,403
10,081	Crohn's disease	186,390	227,023	326,164	197,405
10,089	Crohn's disease	347,786	456,655	544,660	386,430
10,090	Crohn's disease	412,253	337,503	306,485	305,576
10,094	Crohn's disease	256,814	320,654	593,541	406,523
10,095	Crohn's disease	45,568	76,182	27,736	37,539
10,102	Crohn's disease	206,488	236,594	325,721	249,229
10,051	No digestive disease	288,495	266,679	968,229	284,006
10,052	No digestive disease	135,704	180,348	303,864	112,295
10,053	No digestive disease	372,457	545,551	640,635	295,387
10,054	No digestive disease	246,375	279,630	318,118	198,859
10,059	Anal fissure	218,307	224,109	317,736	179,601
10,066	Proctitis/Psoriasis	495,388	657,131	736,683	485,492
10,080	No digestive disease	308,524	445,061	649,226	299,570
10,082	No digestive disease	96,297	150,924	193,102	88,789
10,003	Ulcerative colitis	76,240	126,755	143,652	78,004
10,020	Ulcerative colitis	319,940	583,244	178,132	321,496
10,022	Ulcerative colitis	188,916	399,670	208,178	182,508
10,023	Ulcerative colitis	118,896	136,478	201,303	131,550
10,024	Ulcerative colitis	139,853	233,051	359,658	170,812
10,030	Ulcerative colitis	127,271	136,718	323,945	126,499
10,039	Ulcerative colitis	183,846	235,006	449,717	181,355
10,040	Ulcerative colitis	113,511	179,403	249,464	117,935
10,044	Ulcerative colitis	194,884	169,474	449,998	213,189
10,050	Ulcerative colitis	239,071	305,609	324,797	312,988
10,065	Ulcerative colitis	255,302	270,165	568,720	250,785
10,069	Ulcerative colitis	204,935	197,255	419,431	198,088
10,072	Ulcerative colitis	286,381	174,449	263,180	169,568
10,079	Ulcerative colitis	569,214	361,508	887,360	422,569
10,084	Ulcerative colitis	442,837	206,449	182,069	254,076
10,086	Ulcerative colitis	137,226	98,580	117,532	134,968
10,087	Ulcerative colitis	194,669	142,531	298,763	196,730
10,096	Ulcerative colitis	398,374	513,371	588,895	405,529
10,097	Ulcerative colitis	245,148	235,651	467,802	285,631
	Avarage
	Crohn's disease	405,469	439,668	622,644	444,187
	No Chrohn's Disease	244,372	276,066	400,377	225,862
	Median
	Crohn's disease	341,464	384,850	549,025	370,672
	No Chrohn's Disease	218,307	233,051	323,945	198,088
	ttest CD vs Non CD	0.004639777	0.001853597	0.007398896	0.001413793

TABLE 4


Fluorescent signals from binding of IgM antibodies to different glycans in CD
patients and non CD patients. Glycans are presented in LINEARCODE ®.

Patient
No.	Clinical condition	IgM A[3S]b	IgM Aa	IgM Aa3Ab4GNb	IgM Aa4Ab4Gb	IgM Ab3(GNb6)ANa

10,001	Crohn's disease	0	0	0	0	0
10,004	Crohn's disease	0	0	0	0	0
10,005	Crohn's disease	0	0	0	0	161,405
10,006	Crohn's disease	0	68,568	0	402,561	166,017
10,007	Crohn's disease	0	0	0	0	0
10,008	Crohn's disease	0	0	0	0	0
10,009	Crohn's disease	0	0	0	0	0
10,011	Crohn's disease	0	0	0	0	0
10,012	Crohn's disease	0	0	0	0	0
10,013	Crohn's disease	1,274,136	1,194,898	1,392,444	0	0
10,015	Crohn's disease	387,307	390,644	325,647	0	0
10,016	Crohn's disease	280,897	0	14,251	0	0
10,018	Crohn's disease	248,996	228,314	158,547	0	0
10,021	Crohn's disease	762,735	852,490	700,041	0	327,738
10,025	Crohn's disease	466,121	319,573	565,269	0	0
10,026	Crohn's disease	0	0	0	0	0
10,027	Crohn's disease	151,387	437,186	547,822	0	0
10,028	Crohn's disease	510,204	540,836	723,234	0	81,777
10,031	Crohn's disease	165,011	242,183	199,166	0	15,958
10,033	Crohn's disease	14,440	0	1,113,845	0	0
10,034	Crohn's disease	770,101	695,338	641,641	0	121,757
10,036	Crohn's disease	163,646	642,487	2,180,044	0	0
10,041	Crohn's disease	123,889	89,104	151,581	0	0
10,042	Crohn's disease	0	1,345,482	1,158,234	0	0
10,043	Crohn's disease	0	0	0	0	0
10,047	Crohn's disease	831,510	857,115	1,076,947	—	465,551
10,058	Crohn's disease	128,555	220,493	428,347	—	—
10,060	Crohn's disease	363,862	1,264,367	474,206	—	19,714
10,061	Crohn's disease	690,511	1,095,509	1,128,863	—	621,675
10,062	Crohn's disease	715,200	1,485,943	2,464,680	—	220,921
10,064	Crohn's disease	245,487	664,556	1,633,864	—	635,144
10,067	Crohn's disease	222,329	141,266	75,592	—	—
10,068	Crohn's disease	0	0	0	0	0
10,071	Crohn's disease	67,858	77,830	88,393	0	272,031
10,073	Crohn's disease	0	0	147,447	30,339	184,079
10,074	Crohn's disease	0	0	0	0	0
10,075	Crohn's disease	65,267	34,147	241,365	0	65,722
10,077	Crohn's disease	0	214,916	88,848	0	321,513
10,078	Crohn's disease	0	0	0	0	0
10,081	Crohn's disease	0	0	0	0	0
10,089	Crohn's disease	0	0	0	0	0
10,090	Crohn's disease	0	0	0	0	6,781
10,094	Crohn's disease	68,165	118,072	97,315	93,394	70,477
10,095	Crohn's disease	0	0	0	0	0
10,102	Crohn's disease	0	0	0	0	0
10,051	No digestive disea	0	0	0	0	0
10,052	No digestive disea	0	0	0	0	0
10,053	No digestive disea	0	803,031	314,948	0	0
10,054	No digestive disea	0	0	33,896	86,151	118,374
10,059	Anal fissure	0	0	0	47,680	0
10,066	Proctitis/Psoriasis	255,814	613,172	366,385	711,585	672,896
10,080	No digestive disea	—	142,813	1,437,670	357,136	255,635
10,082	No digestive disea	—	—	—	361,634	265,794
10,003	Ulcerative colitis	—	—	—	—	—
10,020	Ulcerative colitis	—	—	—	—	—
10,022	Ulcerative colitis	0	0	230,936	5,354	0
10,023	Ulcerative colitis	0	0	0	538,071	0
10,024	Ulcerative colitis	0	0	0	119,591	2,544,871
10,030	Ulcerative colitis	0	0	0	0	79,886
10,039	Ulcerative colitis	0	0	0	0	26,754
10,040	Ulcerative colitis	0	0	0	0	0
10,044	Ulcerative colitis	7,575	99,214	210,443	53,421	548,458
10,050	Ulcerative colitis	0	0	0	0	0
10,065	Ulcerative colitis	646,009	651,393	679,823	669,033	381,610
10,069	Ulcerative colitis	0	0	0	0	0
10,072	Ulcerative colitis	0	0	0	0	375,381
10,079	Ulcerative colitis	0	79,891	36,805	512,305	182,972
10,084	Ulcerative colitis	0	0	0	0	0
10,086	Ulcerative colitis	16,235	221,934	0	293,278	0
10,087	Ulcerative colitis	175,021	175,678	321,514	145,748	337,665
10,096	Ulcerative colitis	—	—	—	—	—
10,097	Ulcerative colitis	—	—	—	—	1,022,582
	Avarage
	Crohn's disease	193,725	294,251	395,947	11,695	83,517
	No Crohn's Disea	40,765	103,227	134,534	144,481	252,329
	Median
	Crohn's disease	14,440	68,568	88,848	—	—
	No Crohn's Disea	—	—	—	—	—
	ttest CD vs Non C	0.014433781	0.036400897	0.041051138	0.000390828	0.047775166

Patient No.	Clinical condition	IgM Ab3ANa	IgM GNb3Ab4Gb	IgM GNb3ANa	IgM Dextran	IgM Mannan

10,001	Crohn's disease	0	608,457	657,592	230,160	2,084,216
10,004	Crohn's disease	0	542,436	730,879	360,390	722,969
10,005	Crohn's disease	0	1,375,070	2,933,445	388,048	117,931
10,006	Crohn's disease	0	1,695,894	1,963,416	3,110,617	810,502
10,007	Crohn's disease	0	1,178,646	993,402	969,884	612,220
10,008	Crohn's disease	0	735,201	1,372,932	468,089	796,727
10,009	Crohn's disease	0	322,451	680,970	59,317	0
10,011	Crohn's disease	0	205,150	1,039,057	494,848	0
10,012	Crohn's disease	0	972,528	1,688,071	1,221,314	1,490,590
10,013	Crohn's disease	0	1,174,565	2,419,276	2,049,830	2,588,785
10,015	Crohn's disease	0	1,054,492	1,546,327	2,107,182	1,418,006
10,016	Crohn's disease	0	1,328,058	3,127,245	1,667,531	70,238
10,018	Crohn's disease	0	1,430,548	1,912,812	2,166,938	1,971,045
10,021	Crohn's disease	4,353	1,469,658	2,142,999	2,094,545	1,285,376
10,025	Crohn's disease	0	2,975,994	3,777,466	2,312,303	2,435,341
10,026	Crohn's disease	0	745,061	3,384,432	3,509,606	409,591
10,027	Crohn's disease	0	1,779,829	3,426,917	1,720,479	3,144,625
10,028	Crohn's disease	46,618	1,562,488	2,857,289	2,236,531	3,049,138
10,031	Crohn's disease	0	578,140	849,934	973,540	910,393
10,033	Crohn's disease	0	879,981	1,786,910	822,422	587,545
10,034	Crohn's disease	78,717	1,117,458	1,646,989	1,616,572	1,320,077
10,036	Crohn's disease	0	2,573,605	2,518,175	2,570,459	1,552,108
10,041	Crohn's disease	0	781,745	1,733,620	929,763	1,789,860
10,042	Crohn's disease	0	2,298,533	3,652,328	4,851,471	1,342,805
10,043	Crohn's disease	0	943,254	3,801,228	349,534	628,039
10,047	Crohn's disease	147,112	1,854,934	3,495,774	3,233,236	1,936,982
10,058	Crohn's disease	—	897,206	1,775,488	766,028	983,376
10,060	Crohn's disease	—	926,098	2,910,649	1,296,914	859,571
10,061	Crohn's disease	149,877	2,612,378	3,589,958	2,379,098	4,685,631
10,062	Crohn's disease	188,832	1,464,405	2,716,333	1,256,919	1,245,680
10,064	Crohn's disease	—	1,893,522	3,343,233	2,212,175	2,923,034
10,067	Crohn's disease	—	631,443	1,765,862	1,280,499	1,011,954
10,068	Crohn's disease	0	0	829,715	0	0
10,071	Crohn's disease	0	669,203	1,023,200	302,307	2,573,082
10,073	Crohn's disease	5,781	693,896	1,80,873	1,506,812	2,148,575
10,074	Crohn's disease	0	1,549,121	2,082,886	1,385,468	531,246
10,075	Crohn's disease	0	839,403	1,814,627	1,571,440	582,384
10,077	Crohn's disease	0	676,897	1,309,100	1,059,111	359,244
10,078	Crohn's disease	0	43,955	952,620	464,210	791,441
10,081	Crohn's disease	0	28,386	907,289	0	93,410
10,089	Crohn's disease	0	0	319,608	309,448	0
10,090	Crohn's disease	0	326,922	551,737	253,387	635,071
10,094	Crohn's disease	4,567	427,222	634,599	741,918	1,331,605
10,095	Crohn's disease	0	331,811	1,357,109	7,11,417	2,803,494
10,102	Crohn's disease	0	0	78,782	0	860,022
10,051	No digestive disea	0	0	0	70,898	0
10,052	No digestive disea	0	0	0	0	0
10,053	No digestive disea	0	909,819	1,957,699	2,693,154	160,549
10,054	No digestive disea	0	1,417,516	2,034,745	1,190,821	0
10,059	Anal fissure	0	621,074	860,869	553,501	0
10,066	Proctitis/Psoriasis	648,481	1,399,015	2,112,942	2,036,326	2,851,646
10,080	No digestive disea	13,016	682,951	1,763,399	466,998	226,598
10,082	No digestive disea	58,052	1,059,304	3,803,966	460,919	—
10,003	Ulcerative colitis	—	—	—	—	—
10,020	Ulcerative colitis	—	—	—	—	—
10,022	Ulcerative colitis	0	530,365	1,220,301	910,810	0
10,023	Ulcerative colitis	0	345,812	630,726	401,625	109,462
10,024	Ulcerative colitis	64,642	1,271,762	2,775,365	877,756	1,161,681
10,030	Ulcerative colitis	60,684	606,210	622,605	1,558,797	0
10,039	Ulcerative colitis	32,355	732,277	910,699	542,797	51,325
10,040	Ulcerative colitis	0	798,350	1,289,960	751,879	156,868
10,044	Ulcerative colitis	59,039	402,433	873,504	961,761	697,203
10,050	Ulcerative colitis	0	445,848	728,257	737,999	134,037
10,065	Ulcerative colitis	201,783	1,203,436	1,975,174	1,363,891	1,042,417
10,069	Ulcerative colitis	0	1,127,666	1,331,796	0	313,657
10,072	Ulcerative colitis	0	825,856	1,084,765	2,081,853	566,681
10,079	Ulcerative colitis	79,189	828,413	1,477,648	992,691	696,436
10,084	Ulcerative colitis	0	650,663	1,075,158	309,983	489,824
10,086	Ulcerative colitis	0	835,147	1,931,046	1,405,512	824,630
10,087	Ulcerative colitis	220,927	592,753	1,540,709	897,343	1,016,898
10,096	Ulcerative colitis	—	331,000	441,228	452,374	582,057
10,097	Ulcerative colitis	33,778	854,116	2,517,741	913,480	517,995
	Avarage
	Crohn's disease	13,908	1,024,356	1,895,124	1,333,594	1,277,643
	No Crohn's Disea	54,516	684,140	1,294,463	838,279	429,258
	Median
	Crohn's disease	—	897,206	1,765,852	1,221,314	983,376
	No Crohn's Disea	—	682,951	1,220,301	751,879	160,549
	ttest CD vs Non C	0.059924749	0.029702213	0.17590683	0.034573847	0.000253081

TABLE 5


Specificity and sensitivity of the different IgG anti glycans for differentiation between
CD and other digestive diseases using different cut-off values. The cutoff values for each glycans
where set as the 89 percentiles of the other digestive disease group. Glycans are presented in
LINEARCODE ®.

Anti Glycan IgG antibodies

									M	X
Cut-off level	Gb	Gb3Gb	Gb4Gb	GGN[6S]b	Ma	Ma3(Ma6)Mb	Ma3Ma	Mannan	Xylan	Ma2Ma

65 percentile of	Sensitivity for CD %	76	73	62	62	58	60	71	78	58	71
non CD
	Specificity for CD %	70	63	67	70	67	78	70	67	63	67
75	Sensitivity	62	71	58	60	71	60	64	73	62	71
percentile of non	for CD %
CD
	Specificity	74	74	78	78	52	78	78	74	74	78
	for CD %
85	Sensitivity	56	64	51	49	49	56	62	67	40	62
percentile of non	for CD %
CD
	Specificity	81	81	81	81	85	85	81	81	81	85
	for CD %
90	Sensitivity	49	62	33	42	44	56	60	47	36	40
percentile of non	for CD %
CD
	Specificity	89	89	89	89	89	93	89	89	89	89
	for CD %

TABLE 6


The sensitivity, specificity, True Positives (TP), True Negative (TN), False
Positives (FP), and False Negatives (FN), and Positive Predictive Value
(PPV) in different cut-of values for differentiation between CD and other
digestive disease according to the level of Anti-Glc (β 1-3) Glc (β) IgG.

IgG Gb3Gb
(abnormals
above cut-off)	Sensitivity	Specificity	TP	TN	FP	FN	PPV

—	100.0%	0.0%	45	0	27	0	62.5
0	95.6%	0.0%	43	0	27	2	61.43
6,963	95.6%	3.7%	43	1	26	2	62.32
27,235	93.3%	3.7%	42	1	26	3	61.76
27,402	91.1%	3.7%	41	1	26	4	61.19
62,662	88.9%	3.7%	40	1	26	5	60.61
78,554	88.9%	7.4%	40	2	25	5	61.54
86,949	88.9%	11.1%	40	3	24	5	62.5
87,267	86.7%	11.1%	39	3	24	6	61.9
108,535	86.7%	14.8%	39	4	23	6	62.9
133,683	86.7%	18.5%	39	5	22	6	63.93
156,547	86.7%	22.2%	39	6	21	6	65
174,695	86.7%	25.9%	39	7	20	6	68.1
241,622	84.4%	25.9%	38	7	20	7	65.52
242,565	84.4%	29.6%	38	8	19	7	66.67
312,940	84.4%	33.3%	38	9	18	7	67.86
317,476	84.4%	37.0%	38	10	17	7	69.09
344,750	82.2%	37.0%	37	10	17	8	68.52
371,507	80.0%	37.0%	36	10	17	9	67.92
371,648	80.0%	40.7%	36	11	16	9	69.23
378,722	77.8%	40.7%	35	11	16	10	68.63
379,003	77.8%	44.4%	35	12	15	10	70
430,129	77.8%	48.1%	35	13	14	10	71.43
441,239	77.8%	51.9%	35	14	13	10	72.92
454,736	75.6%	51.9%	34	14	13	11	72.34
489,733	75.6%	55.6%	34	15	12	11	73.91
525,203	73.3%	55.6%	33	15	12	12	73.33
526,443	73.3%	59.3%	33	16	11	12	75
546,432	73.3%	63.0%	33	17	10	12	76.74
612,367	73.3%	66.7%	33	18	9	12	78.57
851,962	73.3%	70.4%	33	19	8	12	80.49
979,509	71.1%	70.4%	32	19	8	13	80
1,209,892	71.1%	74.1%	32	20	7	13	82.05
1,266,954	71.1%	77.8%	32	21	6	13	84.21
1,317,083	68.9%	77.8%	31	21	6	14	83.78
1,376,957	66.7%	77.8%	30	21	6	15	83.33
1,379,223	66.7%	81.5%	30	22	5	15	85.71
1,425,185	64.4%	81.5%	29	22	5	16	85.29
1,461,919	64.4%	85.2%	29	23	4	16	87.88
1,560,904	62.2%	85.2%	28	23	4	17	87.5
1,574,353	62.2%	88.9%	28	24	3	17	90.32
1,705,604	60.0%	88.9%	27	24	3	18	90
1,722,429	57.8%	88.9%	26	24	3	19	89.66
1,732,725	57.8%	92.6%	26	25	2	19	92.86
1,817,947	55.6%	92.6%	25	25	2	20	92.59
1,825,768	53.3%	92.6%	24	25	2	21	92.31
1,869,774	51.1%	92.6%	23	25	2	22	92
1,880,984	48.9%	92.6%	22	25	2	23	91.67
1,994,899	46.7%	92.6%	21	25	2	24	91.3
2,067,775	44.4%	92.6%	20	25	2	25	90.91
2,154,175	42.2%	92.6%	19	25	2	26	90.48
2,324,221	40.0%	92.6%	18	25	2	27	90
2,467,429	37.8%	92.6%	17	25	2	28	89.47
2,551,776	35.6%	92.6%	16	25	2	29	88.89
2,703,085	35.6%	96.3%	16	26	1	29	94.12
2,850,072	33.3%	96.3%	15	26	1	30	93.75
3,096,078	31.1%	96.3%	14	26	1	31	93.33
3,186,273	28.9%	96.3%	13	26	1	32	92.86
3,441,678	28.9%	100.0%	13	27	0	32	100
3,511,076	26.7%	100.0%	12	27	0	33	100
3,559,430	24.4%	100.0%	11	27	0	34	100
3,578,889	22.2%	100.0%	10	27	0	35	100
4,137,076	20.0%	100.0%	9	27	0	36	100
4,327,530	17.8%	100.0%	8	27	0	37	100
5,107,549	15.6%	100.0%	7	27	0	38	100
5,545,432	13.3%	100.0%	6	27	0	39	100
5,640,050	11.1%	100.0%	5	27	0	40	100
5,724,798	8.9%	100.0%	4	27	0	41	100
6,708,583	6.7%	100.0%	3	27	0	42	100
6,891,638	4.4%	100.0%	2	27	0	43	100
7,209,245	2.2%	100.0%	1	27	0	44	100
7,299,442	0.0%	100.0%	0	27	0	45	#####

TABLE 7


Fluorescent signals from binding of IgG antibodies to different glycans in CD Colitis patients and UC patients. Glycans are
presented in LINEARCODE ®.

Patient
No.	Clinical condition	IgG_Aa	IgG_Ab4GNa	IgG_Ab4GNb	IgG_ANa	IgG_Ga

10015	Crohn's disease colitis	2313399.5	635468	690377.5	1472394.5	1738460.25
10018	Crohn's disease colitis	1282598	1535296.5	773145	1304881	1556959.75
10028	Crohn's disease colitis	400412	2027543.5	276464.5	565620.5	933864
10068	Crohn's disease colitis	236509.5	326480.5	365799.5	468415	1037833.75
10089	Crohn's disease colitis	6235212	873746.5	828641.5	879861.5	1551637.75
10102	Crohn's disease colitis	425568	1252680.5	275934.5	402232.5	1119215.5
10105	Crohn's disease colitis
10003	Ulcerative colitis	187885.5	377841	259127	257702	659132.75
10020	Ulcerative colitis	1107251	1374336.5	775070.5	758803.5	1269500.5
10022	Ulcerative colitis	612362	549887	517309	377725.5	1467537
10023	Ulcerative colitis	230639.5	287362	216928.5	159075	693799.5
10024	Ulcerative colitis	984300.5	860812.5	654889	803682	1278164.75
10030	Ulcerative colitis	218405	948666.5	148121.5	131947	660381
10039	Ulcerative colitis	535525.5	355678.5	308129	335650.5	694135
10040	Ulcerative colitis	364266	313945	301998.5	236382	487735.75
10044	Ulcerative colitis	702426.5	809831	486338	678997	1026423.25
10050	Ulcerative colitis	416618	1388773	491837	637010.5	992490
10065	Ulcerative colitis	433662	500898.5	456155	593461	953378.25
10069	Ulcerative colitis	205728.5	714726	259398.5	329163	1215249.5
10072	Ulcerative colitis	192896	194102.5	182286	231396.5	1015614.25
10079	Ulcerative colitis	150330	519316	199636	460673.5	983715.25
10084	Ulcerative colitis	438643	696196	312418	448064	735412.25
10086	Ulcerative colitis	203076.5	203979	127167.5	323691.5	877230.25
10087	Ulcerative colitis	254433.5	421556	304673.5	338759.5	578500.5
10096	Ulcerative colitis	384127	1044393	244746	459440	1205875.25
10097	Ulcerative colitis	174679	267596.5	167481.5	344388.5	751111.75
10029	Ulcerative colitis
Average	Crohn's disease colitis	1,815,617	1,108,536	535,060	848,901	1,322,995
	Ulcerative colitis	410,382	622,631	337,564	416,106	923,441
Median	Crohn's disease colitis	854,083	1,063,214	528,089	722,741	1,335,427
	Ulcerative colitis	364,266	519,316	301,999	344,389	953,378
	ttest	0.012022185	0.026037602	0.044509262	0.002568067	0.006995736

Patient No.	Clinical condition	IgG_Gb	IgG_GN[6S]b	IgG_GNb	IgG_GNb6ANa

10015	Crohn's disease colitis	2507309.25	1752931	2155136.5	2105251
10018	Crohn's disease colitis	2349611.5	1316616	1853996.5	1063920.5
10028	Crohn's disease colitis	527070	842969	815951	1646003
10068	Crohn's disease colitis	646782.25	820937.5	995101.5	1045720
10089	Crohn's disease colitis	3140987	1459663	6756773.5	2984861.5
10102	Crohn's disease colitis	602214.25	684982	984931	788111
10105	Crohn's disease colitis	208854.5
10003	Ulcerative colitis	905251.25	729287	575919	821537.5
10020	Ulcerative colitis	1354221.5	1258840	1372903	1524422.5
10022	Ulcerative colitis	971547.25	805564.5	892125	2107553
10023	Ulcerative colitis	476804.75	335428	380898.5	412904.5
10024	Ulcerative colitis	1536704.5	1278711	1293243.5	1428660.5
10030	Ulcerative colitis	313801.5	288801	368437.5	1521634
10039	Ulcerative colitis	788940	579956.5	603719	900021.5
10040	Ulcerative colitis	508502.25	390982.5	943099.5	1502139
10044	Ulcerative colitis	1134152	794829	1069004	740931
10050	Ulcerative colitis	1307946.75	912013	1120248	836121
10065	Ulcerative colitis	983242.75	842731.5	2518317	810962.5
10069	Ulcerative colitis	598735.5	672092	899127	584783.5
10072	Ulcerative colitis	320461	363892.5	423833	310437.5
10079	Ulcerative colitis	405166	513526.5	508108	411685.5
10084	Ulcerative colitis	703594	545286	826299.5	552446.5
10086	Ulcerative colitis	686424.75	465633	744875	321304
10087	Ulcerative colitis	615110	386386.5	353199	256942.5
10096	Ulcerative colitis	997504	673275	910091.5	558828.5
10097	Ulcerative colitis	424300.25	458307	1859201.5	433687
10029	Ulcerative colitis	1024750
Average	Crohn's disease colitis	1,628,996	1,146,333	2,260,315	1,605,645
	Ulcerative colitis	791,179	647,134	929,613	833,526
Median	Crohn's disease colitis	1,498,197	1,079,793	1,424,549	1,354,962
	Ulcerative colitis	703,594	579,957	892,125	610,963
	ttest	0.009487981	0.003042923	0.022705775	0.012278151

Patient No.	Clinical condition	IgG_Ma	IgG_Ma2Ma	IgG_Ma3(Ma6)Mb	IgG_Ma3Ma	IgG_Mannan

10015	Crohn's disease colitis	1210171.5	1865891	1280893.5	1706919	8298001.25
10018	Crohn's disease colitis	1642422.5	2114538.5	857700.5	1424193	2438472
10028	Crohn's disease colitis	471600	388120	371961.5	644191	1133383.25
10068	Crohn's disease colitis	1065364	1364558	2089715	1525952	856924.5
10089	Crohn's disease colitis	1732145.5	1975468	812187	1751178	1948648.25
10102	Crohn's disease colitis	768939	999741.5	650448	612321	779630.5
10105	Crohn's disease colitis					2577209
10003	Ulcerative colitis	432922	640303	336989	349314.5	1022364.25
10020	Ulcerative colitis	1363896	641830.5	902559	705622.5	2556959.5
10022	Ulcerative colitis	696491.5	689169.5	556384	356351	5619843.5
10023	Ulcerative colitis	741737.5	524735.5	185491	265536	708232.5
10024	Ulcerative colitis	981713	1501423	422316	709226.5	6380358.5
10030	Ulcerative colitis	383159	465097.5	267131	302649	1539392
10039	Ulcerative colitis	427352.5	962878.5	484028.5	500544	1614748.75
10040	Ulcerative colitis	387998	1617762	417664.5	292485	2881787.75
10044	Ulcerative colitis	1166622	830608.5	749019	912334.5	1385639.5
10050	Ulcerative colitis	1056914.5	1200920	756150.5	951103	1829812.5
10065	Ulcerative colitis	666534.5	1165935.5	672182	852588	2333879.75
10069	Ulcerative colitis	359555	599937.5	339385	366514	1736634.75
10072	Ulcerative colitis	455587.5	482990.5	392522	506615	3309624.75
10079	Ulcerative colitis	425263.5	293342.5	370064	356484.5	1665868.75
10084	Ulcerative colitis	592992.5	509651.5	431486.5	430193	1655823.75
10086	Ulcerative colitis	402319	476814.5	379368.5	356194	2174928.5
10087	Ulcerative colitis	409432	380168.5	329778.5	512019.5	1319329
10096	Ulcerative colitis	633246	685412	502613.5	613755	5289044.5
10097	Ulcerative colitis	267456	422519.5	386277	295685	1478518.5
10029	Ulcerative colitis					512254
Average	Crohn's disease colitis	1,148,440	1,451,386	1,010,484	1,277,459	2,575,843
	Ulcerative colitis	623,747	741,658	467,443	507,117	2,447,510
Median	Crohn's disease colitis	1,137,768	1,615,225	834,944	1,475,073	1,541,016
	Ulcerative colitis	455,588	640,303	417,665	430,193	1,736,635
	ttest	0.004574538	0.003038408	0.001689271	2.23605E−05	0.88985599

TABLE 8


Fluorescent signals from binding of IgA antibodies to different glycans in CD Colitis patients and UC patients. Glycans are presented in
LINEARCODE ®.

Patient No.	Clinical condition	IgA_Aa3Ab4GNb3Ab4Gb	IgA_Aa4Ab4Gb	IgA_Ab	IgA_Ab3(GNb6)ANa	IgA_Ab3GNb	IgA_Ab6Ab	IgA_ANa	IgA_ANb

10015	Crohn's disease	823920.5	642431	671617	582327.5	1250743.5	644176	712552.5	782432
10018	Crohn's disease	1120640	692655.5	905785	806576	555300	944644	509272.5	534881
10028	Crohn's disease	70782.5	55409	54145	50555	64293	99547	102094.5	100331.5
10068	Crohn's disease	485132	423765	323220.5	300755	278180.5	314013	335295	303011.5
10089	Crohn's disease	386429.5	475156	347786	300281.5	396131	456654.5	426221.5	420382.5
10102	Crohn's disease	249229	289752.5	206488	157775.5	189939	236594	224709.5	254464
10105	Crohn's disease
10003	Ulcerative colitis	78003.5	74646.5	76239.5	77832.5	76386	128755	99557	98065.5
10020	Ulcerative colitis	321496	266915	319939.5	316325	358795.5	583244	550607	531219.5
10022	Ulcerative colitis	182507.5	253611.5	188916	269222.5	230976.5	399670	278082.5	323070.5
10023	Ulcerative colitis	131549.5	113203.5	118695.5	106896	121660.5	136477.5	183293.5	145928.5
10024	Ulcerative colitis	170812	191188.5	139852.5	313233	141818.5	233050.5	146876	189008.5
10030	Ulcerative colitis	126498.5	114379.5	127270.5	101520.5	93169.5	136717.5	136593.5	231126
10039	Ulcerative colitis	181355	168185	183845.5	185994.5	165022.5	236006	180943	168373
10040	Ulcerative colitis	117934.5	122886.5	113510.5	116982.5	110072.5	176402.5	148789	177785.5
10044	Ulcerative colitis	213188.5	235379.5	194883.5	299006	282039.5	169473.5	153085.5	161501
10050	Ulcerative colitis	312988	253641	239070.5	233244.5	213519.5	305608.5	264615.5	271901
10065	Ulcerative colitis	250784.5	555877.5	255301.5	267297.5	174792	270164.5	251877.5	203122.5
10069	Ulcerative colitis	198087.5	171640.5	204934.5	161723.5	230896.5	197254.5	183682	212249.5
10072	Ulcerative colitis	169567.5	230948	286381	174653	174006	174449	167844	185039.5
10079	Ulcerative colitis	422569	430651.5	569214	337161	328102	361507.5	352796.5	366925
10084	Ulcerative colitis	254076	234589	442836.5	212473	202150.5	206448.5	192001	206100.5
10086	Ulcerative colitis	134968	117207	137226	73241.5	78799.5	98580	67717	101404
10087	Ulcerative colitis	196730	404271.5	194668.5	134105.5	164685	142530.5	145004	171203
10096	Ulcerative colitis	405529	392063.5	398373.5	352667	365977.5	513370.5	391423	433166
10097	Ulcerative colitis	285630.5	280728	245147.5	193135	199541	235651	203575.5	229678
10029	Ulcerative colitis
Average	Crohns	522,689	431,528	418,174	366,378	455,765	449,268	385,024	399,250
	UC	218,646	243,790	233,500	206,659	195,390	247,598	215,703	231,940
Med	Crohns	435,781	449,461	335,504	300,518	337,156	385,334	380,758	361,697
	UC	196,730	234,589	194,884	193,135	174,792	206,449	183,294	203,123
	ttest	0.003432508	0.017087855	0.042789261	0.036632153	0.015545789	0.028930448	0.018529045	0.024586727
	Ulcerative colitis		364,266		519,316	301,999		344,389
	ttest		0.012022185		0.026037602	0.044509262		0.002568067

Patient No.	Clinical condition	IgA_Gb	IgA_Gb3Gb	IgA_GNb	IgA_GNb3Ab4Gb	IgA_GNb3ANa	IgA_GNb4GNb	IgA_GNb6ANa	IgA_Ma

10015	Crohn's disease	2352257.5	3792549.5	1848069.5	957892.25	973773.5	1365558.5	1884673.5	793071.5
10018	Crohn's disease	1744966.5	1050178.5	1108481.5	809108	872211.5	826559	1114615	961742.5
10028	Crohn's disease	193866.5	188673.5	226701.5	144574	153168.25	355502	194085	94336.5
10068	Crohn's disease	1012507	628503	691170.5	1218454	860716.75	920038.25	563962.5	524544
10089	Crohn's disease	993118	1402344.5	975042.5	2982001.5	1030029.75	1025812.25	544660	556490
10102	Crohn's disease	625656	628931.5	317767	288563.75	482396.5	457211.75	325721	261633.5
10105	Crohn's disease				313559	376495	459207.5
10003	Ulcerative colitis	143964	141554.5	200912.5	116158.75	150200.75	168212.25	143651.5	115658.5
10020	Ulcerative colitis	748677	786919.5	923043.5	146937.5	141658	182637	178131.5	256154
10022	Ulcerative colitis	676825	455035	491029.5	218443.5	206741	242902.75	208178	324237.5
10023	Ulcerative colitis	314442	284436.5	275060.5	238630.25	533213	250089	201303	227006.5
10024	Ulcerative colitis	832661.5	442667	479785.5	266009	338630.5	355951.5	359657.5	338794
10030	Ulcerative colitis	409200	284786.5	408023.5	236637.5	206756.5	302015	323944.5	272317.5
10039	Ulcerative colitis	491547	517175.5	392005	786417	809396.5	475749.5	449716.5	387887.5
10040	Ulcerative colitis	279361	218811.5	260669.5	205960.75	169128.25	226448.5	249464	164073.5
10044	Ulcerative colitis	753256	473594.5	555971	392169.25	333466.25	408973.25	449997.5	326666.5
10050	Ulcerative colitis	696130	382399.5	526927.5	383643.5	293206.25	249395.5	324796.5	314891.5
10065	Ulcerative colitis	697130.5	670787.5	457057	291427.75	297671.75	356560.25	568719.5	316328.5
10069	Ulcerative colitis	635934	527218.5	369936.5	194509.5	248548.5	230391	419430.5	389266
10072	Ulcerative colitis	365880.5	357007	390922.5	288597	263714.5	210346.25	263180	229358.5
10079	Ulcerative colitis	554240.5	2257419.5	1048892.5	406795.25	366346	516296.25	887360	514181.5
10084	Ulcerative colitis	581235.5	434302.5	414222	179011.25	203365.25	235661.5	182069	194750.5
10086	Ulcerative colitis	197886.5	207273	193242	433317.5	563802.75	342868	117532	95669.5
10087	Ulcerative colitis	483024	475046	343527	203492.5	517514	190636.5	298763	303731
10096	Ulcerative colitis	669958.5	824120.5	833330.5	445703.25	536801.5	460213	588895	564102
10097	Ulcerative colitis	553467	738068.5	484123	299604.5	305982.25	324637	467802	289141
10029	Ulcerative colitis
Average	Crohns	1,153,729	1,281,530	661,205	1,066,766	728,716	825,114	771,286	531,970
	UC	541,248	551,506	477,300	302,913	341,376	303,578	351,715	296,011
Med	Crohns	1,002,813	839,555	833,107	883,500	866,464	873,299	554,311	540,517
	UC	554,241	455,035	414,222	288,009	297,672	250,089	323,945	303,731
	ttest	0.003969787	0.042899427	0.02591308	0.003184372	0.001126656	1.02649E−05	0.014389095	0.0113452
	Ulcerative colitis	953,378	703,594		579,957		892,125	610,963
	ttest	0.006995736	0.009487981		0.003042923		0.022705775	0.012278151

Patient
No.	Clinical condition	IgA_Ma2Ma	IgA_Ma3(Ma6)Mb	IgA_Ma3Ma	IgA_Ma6Ma	IgA_Mb	IgA_Xa	IgA_Xb	IgA_Mannan	nnan

10015	Crohn's disease	3415595	746825.5	999203.5	988046	1137247	792398.5	989360	1585773.5	1.25
10018	Crohn's disease	2917815.5	727784	717096	884411	1436105	818794.5	701470	1355267.5	8472
10028	Crohn's disease	256032	87390	103552	107441.5	107648	142920	83003.5	861640.5	3.25
10068	Crohn's disease	807816.5	435971	503945.5	812375.5	752123.5	678194	705638.5	1306234.5	24.5
10089	Crohn's disease	819048.5	379526.5	459454	892546.5	1278177.5	674436	787323.5	930450	8.25
10102	Crohn's disease	729964	231998.5	316389	428631	433463	387340.5	392556	899290.5	30.5
10105	Crohn's disease								7209
10003	Ulcerative colitis	287171.5	95306	105407.5	117909	175959.5	155615	153281.5	941585.5	4.25
10020	Ulcerative colitis	1079174.5	192167	234420	483746.5	511744	833230	813730	1227005.5	59.5
10022	Ulcerative colitis	656144	211938	215720.5	361416.5	494957.5	501185	616145	1276257.5	43.5
10023	Ulcerative colitis	637593	207601.5	240763.5	229483.5	248143	288172	261699.5	963954.5	32.5
10024	Ulcerative colitis	1125887.5	262327	295575.5	420147.5	595161.5	517119.5	493560.5	1036056.5	58.5
10030	Ulcerative colitis	866355.5	201701	229526.5	432372	321790.5	320339.5	301633	1372125	392
10039	Ulcerative colitis	710761	276315.5	280618	340776	503526	392243	502137	1239784	8.75
10040	Ulcerative colitis	312896.5	135720	121149.5	177048	234442	270662	236040.5	1052266.5	7.75
10044	Ulcerative colitis	748596	239592.5	308066	337006.5	434514	315294.5	231719.5	731340	39.5
10050	Ulcerative colitis	547079	281154	196723	280097	275211.5	285774	234436	665568.5	12.5
10065	Ulcerative colitis	847658	287586.5	286286.5	285877.5	421455	346017.5	268919	777226.5	9.75
10069	Ulcerative colitis	724998.5	364004	439369	330608	378909.5	415744	300095.5	711639.5	4.75
10072	Ulcerative colitis	817809	179841	253729.5	255584	848373.5	241471.5	238841	1240896	24.75
10079	Ulcerative colitis	1012361	612803	859776.5	425728	618248	513057	575291	993084.5	8.75
10084	Ulcerative colitis	1041362.5	178116	217626	248663.5	316798.5	287812	311632	702478.5	23.75
10086	Ulcerative colitis	191239	121532	100620.5	106336	185590.5	101801.5	98237.5	722440.5	28.5
10087	Ulcerative colitis	700059.5	248842.5	264208.5	266756.5	331533	270612.5	263230	1128040.5	9329
10096	Ulcerative colitis	1394698	522042.5	499770	594745	1301964	613521.5	539601	910216	344.5
10097	Ulcerative colitis	737150	276116	236299	379409	431939.5	331780	311639.5	910626	518.5
10029	Ulcerative colitis
Average	Crohns	1,491,045	434,916	516,807	662,242	857,461	582,347	609,892	1,156,443	2254
	UC	759,415	257,090	283,457	319,716	454,224	368,497	355,467	979,031	843
Med	Crohns	813,433	407,749	481,700	752,461	944,686	676,315	703,554	1,118,342	510
	UC	737,150	239,593	243,764	330,608	421,455	320,340	300,096	963,955	016
	ttest	0.029286279	0.032460361	0.026205708	0.000976406	0.01751963	0.027583859	0.021610873	0.131187019
	Ulcerative colitis	455,588		640,303		417,665		430,193	1,736,635
	ttest	0.004574538		0.003038408		0.001689271		2.23605E−05	0.88985599

TABLE 9


Indication	Tests	Sensitivity, %	Specificity, %

CD vs UC	Anti Mannan	71	91
	(ASCA) (+),
	or
	ALCA (+)
Non-IBD (NIC) vs	ACCA (+),	20	99
IBD (UC + CD)	and
	Anti Mannan
	(ASCA) (−)

Claims

1. A method of diagnosing Crohn's disease in a subject, the method comprising

providing a test sample from said subject; and

determining whether at least one an anti-glycan antibody is present in said sample, wherein said at least one anti-glycan antibody is selected from the group consisting of an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(p antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-Gal 3-sulphate(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-Gal(β) antibody, an anti-GalNAc(α) antibody, an anti-Glc(α) antibody, an anti-Gal(β,1-6)Gal(β) antibody, and an anti-GlcNAc(β,1-6)GalNAc(α) antibody,

wherein the presence of said antibody in said test sample indicates the subject has Crohn's disease.

2. The method of claim 1, wherein said method further comprises comparing the levels of said at least one anti-glycan antibody in said test sample to the levels of said at least one anti-glycan antibody in a control sample, wherein said control sample is selected from the group consisting of one or more individuals known to have or not to have a gastrointestinal disorder other than Crohn's disease.

3. The method of claim 2, wherein said control sample is from one or more individuals with a gastrointestinal disorder that is irritable bowel syndrome or ulcerative colitis.

4. The method of claim 2, wherein said control sample is from one or more individuals that do not have a gastrointestinal disorder.

5. The method of claim 1, wherein said method comprises detecting at least two of said antibodies.

6. The method of claim 1, wherein said method comprises detecting at least four of said antibodies.

7. The method of claim 1, wherein said method comprises detecting at least six of said antibodies.

8. The method of claim 1, further comprising determining whether said test sample has an anti-Mannan (ASCA) antibody, wherein the presence of said anti-ASCA antibody in said sample indicates the subject has Crohn's Disease.

9. The method of claim 1, further comprising determining whether said test sample has an anti-neutrophil cytoplasmic antibodies (ANCA), wherein the presence of said anti-neutrophil cytoplasmic antibodies (ANCA) indicates said subject does not have Crohn's Disease.

10. The method of claim 8, further comprising determining whether said test sample has an anti-neutrophil cytoplasmic antibodies (ANCA), wherein the presence of said anti-neutrophil cytoplasmic antibodies (ANCA) indicates said subject does not have Crohn's Disease.

11. The method of claim 1, wherein said method comprises detecting said anti-Glc(β,1-3)Glc(β) antibody, and one, two, or three of an anti-Man(α,1-3)Man(α) antibody, anti-Man(α,1-3)[Man(α,1-6)]Man(α) antibody, anti-Man(α,1-2)Man(α), anti-Man(α,1-6)Man(α), anti-Mannan (ASCA) antibody or anti-Gal(α) antibody.

12. The method of claim 1, wherein said test sample is a biological fluid.

13. The method of claim 12, wherein said biological fluid is whole blood, serum, plasma, urine, or saliva.

14. The method of claim 11, wherein said wherein said biological fluid is serum.

15. The method of claim 1, further comprising determining the isotype of said antibody.

16. The method of claim 15, wherein said at least one antibody is an IgM type antibody.

17. The method of claim 15, wherein said at least one antibody is an IgA type antibody.

18. The method of claim 15, wherein said at least one antibody is an IgG type antibody.

19. The method of claim 18, wherein said IgG antibody is an anti-Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, or an anti-Xylan antibody.

20. The method of claim 1, wherein said at least one anti-glycan antibody is detected using a fluorescent antibody.

21. The method of claim 1, wherein said at least one anti-glycan antibody is detected using an enzyme-linked immunoabsorbent assay (ELISA).

22. A method of diagnosing Crohn's disease in a subject, the method comprising

providing a test sample from a subject; and

determining whether an anti-glycan antibody is present in said test sample, wherein said at least one anti-glycan antibody is selected from the group consisting of an IgG Glc(β,1-3)Glc(β) antibody and an IgG anti-Man(α,1-3)Man(α) antibody,

wherein the presence of said at least one antibody in said test sample indicates the subject has Crohn's disease.

23. The method of claim 22, wherein said method comprises detecting an IgG anti-Glc(β,1-3)Glc(β) antibody.

24. The method of claim 23, wherein said method comprises detecting an IgG anti-Man(α,1-3)Man(α) antibody.

25. The method of claim 22, wherein said method comprises detecting an IgG Glc(β,1-3)Glc(β) antibody and an IgG anti-Man(α,1-3)Man(α) antibody.

26. The method of claim 22, wherein said method further comprises determining whether said sample has an IgG anti-Mannan or an IgA anti-Mannan antibody, wherein the presence of said IgG anti-Mannan or IgA anti-Mannan antibody in said sample indicates said subject has Crohn's disease.

27. The method of claim 26, wherein said method comprises determining whether said sample has an IgG anti-Mannan antibody.

28. The method of claim 26, wherein said method comprises determining whether said sample has an IgA anti-Mannan antibody.

29. The method of claim 26, wherein said method further comprises determining whether said sample has an anti-neutrophil cytoplasmic antibodies (ANCA), wherein the absence of said antibody in said sample indicates said subject has Crohn's disease.

30. A method of differentially diagnosing Crohn's disease or inflammatory bowel disease in a subject, the method comprising

providing a test sample from a subject; and

determining whether said sample has an antibody selected from the group consisting of an

anti-neutrophil cytoplasmic antibody (ANCA),

IgG anti-Glc(β,1-3)Glc(β)

IgG ASCA; and

IgA ASCA,

wherein the absence of ANCA and the presence of at least one of said IgG anti-Glc(β,1-3)Glc(β) IgG ASCA, and IgA ASCA antibodies in said test sample indicates the subject has Crohn's disease, and

wherein the presence of at least one of said antibodies in said test sample indicates the subject has inflammatory bowel disease (IBD).

31. A method of differentially diagnosing Crohn's disease colitis and ulcerative colitis in a subject, the method comprising

providing a test sample from a subject; and

determining levels of at least one an anti-glycan antibody in said sample, wherein said at least one anti-glycan antibody is selected from the group consisting of

an IgG anti-Gal(α,1-4)GlcNAc(α) antibody,

an IgG anti-Gal(β,1-4)GlcNAc(13) antibody,

an IgG anti-GalNAc(α) antibody,

an IgG anti-Glc(α) antibody,

an IgG anti-Glc(β) antibody,

an IgG anti-GlcNAc(β,6-Sulphate) antibody,

an IgG anti-GlcNAc(β) antibody,

an IgG anti-GlcNAc(β,1-6)GalNAc(α) antibody,

an IgA anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody,

an IgA anti-Gal(α,1-4)Gal(β,1-4), Glc(β) antibody,

an IgA anti-Gal(β) antibody,

an IgA anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody,

an IgA anti-Gal(β,1-3)GlcNAc(β) antibody,

an IgA anti-Gal(β,1-6)Gal(β) antibody,

an IgA anti-GalNAc(α) antibody,

an IgA anti-GalNAc(β) antibody,

IgA an anti-Glc(β) antibody,

an IgA anti-Glc(β,1-3)Glc(β) antibody,

an IgA anti-GlcNAc(β) antibody,

an IgA anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody,

an IgA anti-GlcNAc(β,1-3)GalNAc(α) antibody,

an IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody,

an IgA anti-GlcNAc(β,1-6)GalNAc(α) antibody, and

an IgA anti-Xyl(β) antibody,

wherein the presence of said at least one antibody in said test sample indicates the subject has Crohn's disease colitis.

32. The method of claim 31, wherein said method further comprises comparing the levels of said at least one anti-glycan antibody in said test sample to the levels of said at least one anti-glycan antibody in a control sample, wherein said control sample is selected from the group consisting of one or more individuals known to have or not to have Crohn's disease colitis or known to have or not to have ulcerative colitis (IC).

33. The method of claim 31, further comprising

determining whether an additional anti-glycan antibody anti-glycan antibody is present in said sample, wherein said additional anti-glycan antibody is selected from the group consisting of

an IgG anti-Gal(α) antibody,

an IgG anti-Man(α) antibody,

an IgG anti-Man(α,1-3)Man(α,1-6)Man(β) antibody,

an IgG anti-Man(α,1-3)Man(α) antibody,

an IgA anti-Man(α) antibody,

an IgA anti-Man(α,1-2)Man(α) antibody,

an IgA anti-Man(α,1-3)Man(α,1-6)Man(β) antibody,

an IgA anti-Man(β, 1-3)Man(α) antibody,

an IgA anti-Man(α,1-6)Man(α) antibody,

an IgA anti-Man(β) antibody, and

an IgA anti-X(α) antibody,

wherein the presence of said additional antibody in said test sample indicates the subject has Crohn's disease colitis.

34. The method of claim 31, wherein said at least one antibody is the IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody.

35. The method of claim 33, wherein said additional antibody is the IgG anti-Man(α,1-3)Man(α) antibody.

36. The method of claim 35, wherein said at least one antibody is IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody.

37. The method of claim 31, wherein said method comprises detecting at least two of said antibodies.

38. The method of claim 31, wherein said method comprises detecting at least four of said antibodies.

39. The method of claim 31, wherein said method comprises detecting at least six of said antibodies.

40. The method of claim 31, wherein said test sample is a biological fluid.

41. The method of claim 31, wherein said biological fluid is whole blood, serum, plasma, urine, or saliva.

42. The method of claim 31, wherein said wherein said biological fluid is serum.

43. A method for differentially diagnosing inflammatory bowel disease (IBD) or non-IBD digestive disease (NIC) in a subject, the method comprising

providing a test sample from a subject with symptoms of NIC or IBD;

determining if anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate Antibodies (ACCA) and anti-mannan (ASCA) antibodies are present in said sample,

wherein the presence of ACCA antibodies and the absence of ASCA antibodies in said sample indicates said subject has NIC.

44. The method of claim 43 where the determining if anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate Antibodies (ACCA) and anti-mannan (ASCA) antibodies are present in said sample is done by

comparing levels of said antibodies to levels of antibodies in a reference sample from a subject known to have IBD,

wherein a higher level of ACCA antibodies and a lower level of ASCA antibodies in said test sample relative to the said reference sample indicates said patient has NIC.

45. The method of claim 43, further comprising determining whether said test sample has anti-laminarobioside (Glc(β,1-3)Glc(β)) Carbohydrate Antibodies (ALCA) antibodies, wherein the absence of ALCA antibodies in said sample indicates said subject has NIC.

46. A kit for diagnosing Crohn's Disease, the kit comprising one or more reagents that specifically detect an antibody selected from the group consisting of an anti-Glc(β) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfateantibody, an anti-Man(α,1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-Gal 3-sulphate(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(β) antibody, and an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody.

47. The kit of claim 46, further including instructions for using said kit.

48. The kit of claim 46, wherein said kit further comprises one or more reagents that specifically detect an anti-Mannan (ASCA) antibodies or anti-neutrophil cytoplasmic antibodies (ANCA).

49. The kit of claim 46, wherein said kit comprises a reagent or reagents that specifically detects an anti-Mannan (ASCA) antibody and a anti-neutrophil cytoplasmic antibodies (ANCA).

50. The kit of claim 46, further comprising a reagent that specifically detects an IgG, IgM, or IgA-type antibody.

51. The kit of claim 46, wherein said reagents are provided on an array.

52. The kit of claim 50, wherein said kit comprises reagents that detect IgG Glc(β,1-3)Glc(β) antibody and an IgG anti-Man(α,1-3)Man(α) antibody.

53. A kit for diagnosing Crohn's Disease, the kit comprising one or more reagents that specifically detect an antibody selected from the group consisting of at least one anti-glycan antibody is selected from the group consisting of

an IgG anti-Gal(α,1-4)GlcNAc(α) antibody,

an IgG anti-Gal(β,1-4)GlcNAc(β) antibody,

an IgG anti-GalNAc(α) antibody,

an IgG anti-Glc(α) antibody,

an IgG anti-Glc(β) antibody,

an IgG anti-GlcNAc(β,6-Sulphate) antibody,

an IgG anti-GlcNAc(β) antibody,

an IgG anti-GlcNAc(β,1-6)GalNAc(α) antibody,

an IgA anti-Gal(α,14)Gal(,1-4), Glc(β) antibody,

an IgA anti-Gal(β) antibody,

an IgA anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody,

an IgA anti-Gal(β,1-3)GlcNAc(β) antibody,

an IgA anti-Gal(β,1-6)Gal(β) antibody,

an IgA anti-GalNAc(α) antibody,

an IgA anti-GalNAc(β) antibody,

IgA an anti-Glc(β) antibody,

an IgA anti-Glc(β,1-3)Glc(β) antibody,

an IgA anti-GlcNAc(β) antibody,

an IgA anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody,

an IgA anti-GlcNAc(β,1-3)GalNAc(α) antibody,

an IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody,

an IgA anti-GlcNAc(β,1-6)GalNAc(α) antibody, and

an IgA anti-Xyl(β) antibody.

54. The kit of claim 53, further comprising one or more reagents that specifically detect an antibody selected from the group consisting of at least one anti-glycan antibody is selected from the group consisting of

an IgG anti-Gal(α) antibody,

an IgG anti-Man(α) antibody,

an IgG anti-Man(α,1-3)Man(α,1-6)Man(β) antibody,

an IgG anti-Man(α,1-3)Man(α) antibody,

an IgA anti-Man(α) antibody,

an IgA anti-Man(α,1-2)Man(α) antibody,

an IgA anti-Man(α,1-3)Man(α,1-6)Man(β) antibody,

an IgA anti-Man(β, 1-3)Man(α) antibody,

an IgA anti-Man(α,1-6)Man(α) antibody,

an IgA anti-Man(β) antibody, and

an IgA anti-Xyl(α) antibody.

and IgA anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β) antibody.

an IgA anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody,

55. The kit of claim 54, further including instructions for using said kit.

56. The kit of claim 54, wherein said kit further comprises one or more reagents that specifically detect an anti-Mannan (ASCA) antibodies or anti-neutrophil cytoplasmic antibodies (ANCA).

57. The kit of claim 56, wherein said kit comprises a reagent or reagents that specifically detects an anti-Mannan (ASCA) antibody and anti-neutrophil cytoplasmic antibodies (ANCA).

58. The kit of claim 53, further comprising a reagent that specifically detects an IgG, IgM, or IgA-type antibody.

59. The kit of claim 53, wherein said reagents are provided on an array.

60. A kit for differentially diagnosing Crohn's disease and inflammatory bowel disease in a subject, the kit comprising one or more reagents that detect an antibody selected from the group consisting of an anti-neutrophil cytoplasmic antibody (ANCA), IgG anti-Glc(β,1-3)Glc(β, IgG ASCA; and IgA ASCA.

61. The kit of claim 60, further comprising instructions for using said kit.

62. A kit for differentially diagnosing Crohn's disease colitis and ulcerative colitis in a subject, the kit comprising one or more reagents that detect at least one anti-glycan antibody selected from the group consisting of an IgG anti-Gal(α,14)GlcNAc(α) antibody, an IgG anti-Gal(β,1-4)GlcNAc(β) antibody, an IgG anti-GalNAc(α) antibody, an IgG anti-Glc(α) antibody, an IgG anti-Glc(β) antibody, an IgG anti-GlcNAc(β,6-Sulphate) antibody, an IgG anti-GlcNAc(β) antibody, an IgG anti-GlcNAc(β,1-6)GalNAc(α) antibody, an IgA anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an IgA anti-Gal(α,1-4)Gal(β,1-4), Glc(β) antibody, an IgA anti-Gal(β) antibody, an IgA anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody, an IgA anti-Gal(β,1-3)GlcNAc(β) antibody, an IgA anti-Gal(β,1-6)Gal(β) antibody, an IgA anti-GalNAc(α) antibody, an IgA anti- GalNAc(β) antibody, IgA an anti-Glc(β) antibody, an IgA anti-Glc(β,1-3)Glc(β) antibody, an IgA anti-GlcNAc(β) antibody, an IgA anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an IgA anti-GlcNAc(β,1-3)GalNAc(α) antibody, an IgA anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an IgA anti-GlcNAc(β,1-6)GalNAc(O) antibody, and an IgA anti-Xyl(β) antibody.

63. The kit of claim 62, further comprising instructions for using said kit.

64. A kit for differentially diagnosing inflammatory bowel disease (IBD) or non- IBD digestive disease (MC) in a subject, the kit comprising one or more reagents that detect at least one anti chitobioside (GlcNAc(β,1-4)GlcNAc(β)) carbohydrate Antibodies (ACCA) and anti-mannan (ASCA) antibodies.

65. The kit of claim 64, further comprising instructions for using said kit.

66. An array comprising a plurality of carbohydrate reagents that specifically detect an antibody selected from the group consisting of an anti-Glc(β) antibody, an anti-Gal(α) antibody, an anti-Glc(β,1-4)Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) 6-sulfate antibody, an anti-Man(c, 1-2)Man(α) antibody, an anti-Man(α,1-3)Man(α) antibody, an anti-Man(α,1-6)Man(α) antibody, an anti-Man(α) antibody, an anti-Man(α,1-3)[Man(α,1-6)]Man(α), an anti-Dextran antibody, an anti-Xylan antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-Gal 3-sulphate(p) antibody, an anti-aGlcNAc(β,1-3)GalNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, and an anti-Gal(α,1-3)Gal(β,1-4)GlcNAc(β) antibody, wherein said reagents are attached at an addressable location on said array.

67. The array of claim 66, further comprising a carbohydrate reagent or reagents that detect an anti-Mannan (ASCA) antibody or a anti-neutrophil cytoplasmic antibodies (ANCA).

68. The array of claim 66, wherein said array comprises carbohydrate reagent or reagents that detect an anti-Mannan (ASCA) antibody and anti-neutrophil cytoplasmic antibodies (ANCA).

69. The array of claim 66, wherein each of said glycans are attached to said array via a linker.

70. The array of claim 69, wherein said linker includes at least one ethylene glycol derivative, at least two cyanuric chloride derivatives and an anilino group.

71. The array of claim 66, wherein at least two of said reagent or reagents are provided at the same location on said addressable array.

72. A glycan reagent for diagnosing CD, wherein said reagent specifically detects at least one anti-glycan antibody selected from the group consisting of an anti-Gal(α,1-4)GlcNAc(x) antibody, an anti-Gal(β,1-4)GlcNAc(β) antibody, an anti- GalNAc(α) antibody, an anti-Glc(β) antibody, an anti-Glc(α) antibody, an anti- GlcNAc(β,6-Sulphate) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β,1-6)GalNAc(α) antibody, an anti-Gal(α,1-4)Gal(β,1-4), Glc(β) antibody, an anti-Gal(β) antibody, an anti-Gal(β,1-3)[GlcNAc(β,1-6)]GalNAc(α) antibody, an anti-Gal(β,1-3)GlcNAc(β) antibody, an anti-Gal(0,1-6)Gal(β) antibody, an anti-GalNAc(α) antibody, an anti-GalNAc(β) antibody, an anti-Glc(β) antibody, an anti-Glc(β,1-3)Glc(β) antibody, an anti-GlcNAc(β) antibody, an anti-GlcNAc(β,1-3)Gal(β,1-4)Glc(β) antibody, an anti-GlcNAc(β,1-3)GalNAc(α) antibody, an anti-GlcNAc(β,1-4)GlcNAc(β) antibody, an anti-GlcNAc(β,1-6)GalNAc(α) antibody, an anti-Xyl(β) antibody, and an anti-Xyl(α) antibody.