Patent Application
20110136255
The invention generally relates to methods of identifying Crohn's disease patients who require surgery and/or who are susceptible to developing complications during the course of disease.
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestines of unknown etiology. There are two major types of IBD: Crohn's disease (CD) and ulcerative colitis (UC). The diagnosis of IBD is based on a combination of clinical, endoscopic, histopathological, radiologic and laboratory characteristics.
The clinical course of CD is frequently characterized by complications and the need for surgery. A recent study reported that 65-80% of patients with CD undergo at least one surgery during the course of the disease, while many patients require multiple surgical interventions. The complicated disease phenotype is characterized by the presence of fistulae and/or stenosis. About 30% of patients diagnosed with inflammatory disease develop penetrating disease (characterized by abscess, fistula, or phlegmon) within 10 years. The occurrence of complications such as fistulae, stenosis, and the need for surgery cause significant health care burdens.
Therefore, there is a need to identify CD patients who are at risk of requiring surgery within a certain time frame and/or who are susceptible to developing complications repeatedly during the course of the disease. A series of treatment options/levels (e.g., a treatment pyramid) is available for the treatment of CD, wherein more aggressive treatment (a higher level in the pyramid) is utilized for patients with more complicated CD or for patients with the risk of developing complicated CD. In practice, the existence of risk factors, as well as clinical observations, allows physicians to initiate treatment with more aggressive biological agents or to escalate treatment to a higher/more aggressive level, if necessary. Thus, patients identified with a higher risk for disease complications and surgeries are targeted with more aggressive (higher level in the treatment pyramid) therapeutic management.
The invention is based, in part, on the determination that anti-laminarin (anti-L) and anti-chitin (anti-C) antibodies serve as prognostic indices for Crohn's disease (CD). Qualitative assessment of the presence of anti-C antibodies in a blood sample, e.g., blood serum of an individual, is useful for risk assessment purposes. The presence of anti-chitin antibodies is correlated with the need for surgical intervention. Assessment of anti-C antibodies has been hampered due to difficulties (e.g., poor solubility) in working with the capture reagent, chitin.
Accordingly, the invention provides methods of predicting a risk for complicated course of CD in a subject. First, a blood sample is provided from a subject with symptoms of CD. Second, the levels of a variety of antibodies are detected in the blood sample. The level of an anti-chitin (anti-C) antibody in a blood sample is detected by binding to a carbohydrate reagent comprising an isolated chitosan molecule. The level of an anti-laminarin (anti-L) antibody in the sample is detected by binding to a carbohydrate reagent comprising an isolated laminarin molecule. The level of an anti-mannobioside carbohydrate antibody (AMCA) in the blood sample is detected by binding to a carbohydrate reagent comprising an isolated mannobioside molecule, and the level of an anti-Saccharomyces cerevisiae antibody (gASCA) in the sample is detected by binding to a carbohydrate reagent comprising an isolated mannan. A complicated disease course is predicted by detection of an elevated level of the antibodies in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD. The complicated disease course includes stricturing disease, penetrating disease, or perianal disease. Optionally, an elevated level of the antibodies in the blood sample relative to the reference level or the control sample indicates the subject will need abdominal surgery.
In one aspect, the method of predicting the course of CD in a subject further comprises detecting the levels of additional antibodies in the sample. The level of an anti-laminaribioside carbohydrate antibody (ALCA) in the sample is detected by binding to a carbohydrate reagent comprising an isolated laminaribioside molecule. The level of an anti-chitobioside carbohydrate IgA antibody (ACCA) in the sample is detected by binding to a carbohydrate reagent comprising an isolated chitobioside molecule. A complicated disease course is predicted by detection of an elevated level of the antibodies in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that have do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD. The complicated disease course includes stricturing disease, penetrating disease, or perianal disease. In one aspect; an elevated level of the antibodies in the blood sample relative to the reference level or the control sample indicates the subject will need abdominal surgery.
Other methods of predicting CD in a subject include providing a blood sample from a subject with CD and detecting a level of an anti-chitin (anti-C) antibody in the sample by binding to a carbohydrate reagent comprising an isolated chitosan molecule. A complicated disease course is predicted by detection of an elevated level of the antibody in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD.
Also provided is a reagent suitable for predicting the course of CD in a subject, wherein the reagent comprises an isolated chitosan molecule. The isolated chitosan molecule detects a level of an anti-C antibody. A complicated disease course is predicted by detection of an elevated level of the anti-C antibody relative to a reference level or a control sample from a control population of one or more individuals that have non-complicated CD. In one aspect, the isolated chitosan molecule comprises a chitosan lactate molecule. Optionally, the isolated chitosan lactate molecule is associated with a solid substrate. Preferably, the isolated chitosan lactate molecule is associated with the substrate in the absence of a linker.
Reagents suitable for predicting the course of CD in a subject include those that can detect levels of specific antibodies in the subject. The reagent comprises an isolated chitosan molecule, an isolated laminarin molecule, an isolated mannobioside molecule, and an isolated mannan. The isolated chitosan molecule detects a level of an anti-C antibody, while the isolated laminarin molecule detects a level of an anti-L antibody. Similarly, the isolated mannobioside molecule detects a level of an anti-mannobioside carbohydrate antibody (AMCA), while the isolated mannan molecule detects a level of an anti-Saccharomyces cerevisiae antibody (gASCA). A complicated disease course is predicted by detection of an elevated level of the antibodies relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD.
The reagent suitable for predicting the course of CD in a subject may additionally include an isolated chitobioside molecule and an isolated laminaribioside molecule. The isolated chitobioside molecule detects a level of an anti-chitobioside carbohydrate IgA antibody (ACCA), while the isolated laminaribioside molecule detects a level of an anti-laminaribioside carbohydrate antibody (ALCA). A complicated disease course is predicted by detection of an elevated level of the antibodies relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD.
The isolated antigens are purified or synthetic. By “purified” or “substantially purified” is meant a laminarin, chitin, or chitosan molecule or biologically active portion thereof that is substantially free of cellular material or other contaminating macromolecules, e.g., polysaccharides, nucleic acids, or proteins, from the cell or tissue source from which the laminarin, chitin, or chitosan is derived. The phrase “substantially purified” also includes a laminarin, chitin, or chitosan molecule that is substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of laminarin, chitin, or chitosan that are separated from cellular components of the cells from which they are isolated.
Levels of the antibodies are determined by direct or indirect detection. The amounts of the antibody are determined by measuring binding of the antibody to an isolated synthetic or purified molecule or a molecule attached, optionally via a linker, to a solid phase, e.g., a substrate, a plate, or a chip. Optionally, the levels of the antibodies are determined by binding to a carbohydrate reagent comprising an isolated molecule. Alternatively, the amounts of the antibody are determined by measuring binding of the antibody to a polysaccharide containing the antigen.
By “reference level” is meant the mean, median, or range level of each antibody in a population of individuals that do not have CD. Alternatively, the reference level is the mean level plus two standard deviations (SD) of each antibody in a population of one or more individuals that do not have CD. Optionally, the reference level is a qualitative, not a quantitative reference and is expressed in arbitrary units. The greater the relative antibody level compared to the reference level, the greater the risk for the development of complicated CD. For example, if the reference level is about 50 arbitrary units of anti-C antibody, an anti-C antibody level of about 56 arbitrary units would indicate a risk of complications or surgery; an antibody level of about 75 arbitrary units would indicate a high risk of complications or surgery; and an antibody level of about 100 arbitrary units would indicate a very high risk of complications or surgery.
As used herein, the term “specificity” means the probability that a method is negative in the absence of the measured trait. 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 the measured trait. The antibody cut-off value can be selected such that, when the sensitivity is at least about 60%, the specificity of diagnosing/prognosing an individual is in the range of 80-85%, for example, 85-90%, 90-95%, or 95-99%. Optionally, complicated CD is prognosed if the amount of the antibody is greater than the amount of the antibody in a control sample at a cutoff value providing a specificity in the range of 30-60%, for example, at least about 35-60%, 40-60%, 45-60%, or 50-60%. Alternatively, complicated CD is prognosed if the amount of the antibody is greater than the amount of the antibody in a control sample at a cutoff value providing a specificity of at least about 80%, at least about 85%, at least about 90%, or at least about 95%.
The antibodies described herein are useful for assessing the risk of a complicated disease course or the need for surgery in CD patients. The patients may require surgery within at least about 1 month; at least about 3 months; at least about 6 months; at least about 12 months; at least about 18 months; at least about 20 months; at least about 24 months; at least about 40 months; or at least about 80 months. A complicated disease course or a higher risk of the development of complicated CD is predicted/prognosed by detecting elevated levels of at least one of an anti-L antibody and an anti-C antibody. First, a blood sample is provided from a subject with symptoms of CD. Alternatively, a blood sample is provided from a patient diagnosed with CD. The level of an anti-laminarin (anti-L) antibody in the sample is detected by binding to an isolated laminarin molecule. The level of an anti-chitin (anti-C) antibody in the sample is detected by binding to an isolated chitin molecule or a chitosan/chitosan lactate molecule. Optionally, the levels of the antibodies are determined by binding to a carbohydrate reagent comprising an isolated molecule. A complicated disease course is predicted by detection of an elevated level of the antibodies in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD, e.g., at least 5%, at least 10%, at least 25%, or at least 50% greater, or at least 2 fold, at least 5 fold, or at least 10 fold greater than a reference level or the amount of the antibody in the control sample. The greater the relative antibody level compared to the reference level, the greater the risk for the development of complicated CD. The complicated disease course includes stricturing disease, penetrating disease, or perianal disease. An elevated level of at least one of an anti-L antibody or an anti-C antibody in the blood sample relative to the reference level or the control sample indicates the subject will need abdominal surgery.
The detection of high levels of an anti-L antibody or an anti-C antibody, alone or in conjunction with gASCA, enables the identification of CD patients who may require surgery within a certain time frame and/or who are susceptible to develop complications and may need to undergo surgery repeatedly during the course of the disease. The patients may require surgery within at least about 1 month; at least about 3 months; at least about 6 months; at least about 12 months; at least about 18 months; at least about 20 months; at least about 24 months; at least about 40 months; or at least about 80 months. These patients should be targeted for more aggressive therapeutic management.
A complicated disease course or a higher risk of the development of complicated CD is predicted/prognosed by detecting elevated levels of at least one of an anti-L antibody and an anti-C antibody, and one or more of an anti-Saccharomyces cerevisiae antibody (gASCA), an anti-laminaribioside carbohydrate antibody (ALCA), an anti-chitobioside carbohydrate IgA antibody (ACCA), and an anti-mannobioside carbohydrate antibody (AMCA). More specifically, a complicated disease course or a higher risk of the development of complicated CD is predicted/prognosed by detecting elevated levels of an anti-L antibody, an anti-C antibody, AMCA, and gASCA. Alternatively, a complicated disease course or a higher risk of the development of complicated CD is predicted/prognosed by detecting elevated levels of an anti-L antibody, an anti-C antibody, AMCA, gASCA, ALCA, and ACCA. The level of an anti-laminarin antibody in the sample is detected by binding to an isolated laminarin molecule. The level of an anti-chitin antibody in the sample is detected by binding to an isolated chitin molecule or an isolated chitosan/chitosan lactate molecule. The level of an anti-Saccharomyces cerevisiae antibody (gASCA) in the sample is detected by binding to an isolated mannan. The level of an anti-laminaribioside carbohydrate antibody (ALCA) in the sample is detected by binding to an isolated laminaribioside molecule. The level of an anti-chitobioside carbohydrate IgA antibody (ACCA) in the sample is detected by binding to an isolated chitobioside molecule. The level of an anti-mannobioside carbohydrate antibody (AMCA) in the sample is detected by binding to an isolated mannobioside molecule. Optionally, the levels of the antibodies are determined by binding to a carbohydrate reagent comprising an isolated molecule. A complicated disease course is predicted/prognosed by detection of an elevated level of at least one of anti-L and anti-C, and one or more of gASCA, ALCA, ACCA, or AMCA in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD, e.g., at least 5%, at least 10%, at least 25%, or at least 50% greater, or at least 2 fold, at least 5 fold, or at least 10 fold greater than a reference level or the amount of the antibody in the control sample. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD. The greater the antibody level compared to the reference level, the greater the risk for the development of complicated CD. The complicated disease course includes stricturing disease, penetrating disease, or perianal disease. More specifically, a complicated disease course is predicted/prognosed by detection of an elevated level of an anti-L antibody, an anti-C antibody, AMCA, and gASCA. Alternatively, a complicated disease course is predicted/prognosed by detection of an elevated level of an anti-L antibody, an anti-C antibody, AMCA, gASCA, ALCA, and ACCA.
The course of CD or a higher risk of the development of complicated disease is predicted/prognosed by detecting elevated levels of an anti-L antibody. First, a blood sample is provided from a subject with symptoms of CD. The level of an anti-laminarin (anti-L) antibody in the sample is detected by binding to an isolated laminarin molecule. Optionally, the levels of the antibody are determined by binding to a carbohydrate reagent comprising an isolated molecule. A complicated disease course is predicted by detection of an elevated level of the antibody in the blood sample relative to a control sample or reference level from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD.
The course of CD or a higher risk of the development of complicated disease is predicted/prognosed by detecting elevated levels of an anti-C antibody. First, a blood sample is provided from a subject with symptoms of CD. Alternatively, a blood sample is provided from a patient diagnosed with CD. The level of an anti-chitin (anti-C) antibody in the sample is determined by binding to an isolated chitin molecule or an isolated chitosan/chitosan lactate molecule. Optionally, the levels of the antibodies are determined by binding to a carbohydrate reagent comprising an isolated molecule. A complicated disease course is predicted by detection of an elevated level of the antibody in the blood sample relative to a reference level or a control sample from a control population of one or more individuals that do not have CD or have non-complicated CD. Optionally, the reference level is the level under which exists the antibody level of a majority of individuals that do not have CD or have non-complicated CD, e.g., at least about 80-85%; at least about 85-90%; at least about 90-95%; or at least about 95-99% of individuals with antibody levels below the reference level do not have CD or have non-complicated CD.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, patents, 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.
IBDX® is an immunoassay kit containing capture reagents developed and produced by Glycominds Ltd. for the detection of antibodies against the mannan epitope of Saccharomyces cerevisiae (anti-Saccharomyces cerevisiae antibodies; gASCA), laminaribioside (anti-laminaribioside carbohydrate antibodies; ALCA), chitobioside (anti-chitobioside carbohydrate IgA antibodies; ACCA) and mannobioside (anti-mannobioside carbohydrate antibody; AMCA). The intended use of IBDX® is to aid in the diagnosis of CD patients. In conjunction with anti-neutrophil cytoplasmic antibodies (pANCA), IBDX® can be used to aid in IBD diagnosis and the stratification of IBD patients to CD and UC patients. A higher number of antibodies identified with IBDX®, and a higher level of gASCA, ALCA, ACCA or AMCA antibodies is associated with a more complicated CD disease behavior (presence of strictures or fistula), a higher frequency of abdominal surgery and a steroid refractory response (Dotan I et al., 2006 Gastroenterology, 131:366-378; Ferrante M et al., 2007 Gut, 56:1394-1403; and Papp M et al., 2008 Am J Gastroenterol, 103(3):665-81).
As described herein, unexpectedly, in addition to antibodies detected by IBDX®, the level of anti-laminarin (anti-L) IgA and anti-chitin (anti-C) IgA antibodies in a patient's bloodstream (both individually and combined) serves as a risk indicator/assessment and prognostic index for Crohn's disease. Enzyme immune assay (EIA) methods using the antigens laminarin and chitosan lactate were developed for the detection of anti-L and anti-C levels in sera from human patients.
The invention provides different methods of preparing an antigen, e.g., the small molecules described herein, for recognition by an antibody. In order to bind to an antibody, large quantities of the small molecule, e.g., chitobioside (
Anti-chitobioside carbohydrate antibodies (ACCA) are associated with complicated Crohn's disease behavior. Because chitobioside (
Prior to this invention, the detection of anti-chitobioside antibodies was performed by covalently attaching p-aminophenyl chitobioside to a solid phase. The solid phase was subsequently exposed to patient sera, and the antigen-specific antibodies bound to the antigens on the surface of the solid phase. The bound antibodies were detected and quantified via secondary labeling. However, the process of linker synthesis and the covalent attachment of the linker to chitobioside is expensive and involves many lengthy, complicated steps. As such, prior to the invention described herein, there was a need for the development of a simpler, quicker process for the identification of chitobioside antibodies.
A suitable alternative to chitobioside may include chitin, a linear polymer of β(1,4)-linked N-acetyl-D-glucosamine composed of chitobioside building blocks. See,
As shown in
Chitosan lactate is a water-soluble derivative of chitin; however, given the low structure similarity of chitosan lactate and chitin (10%-20%), one skilled in the art of carbohydrate chemistry would not expect that chitosan lactate would bind to anti-chitin antibody. Surprisingly, as described herein, chitosan lactate was best for detection anti-C antibodies. Chitosan lactate is not expressed on the surface of Saccharomyces cerevisiae. Therefore, ASCA does not bind to these molecules. Moreover, anti-C antibodies recognized chitosan in the absence of a chemical linker attaching the small molecule to a solid phase.
The use of chitosan is a significant improvement over prior methods, because it eliminates the need to covalently attach chitobioside to a linker and allows for the absorption of chitosan directly to a solid phase. Chitosan is used for the detection of anti-C antibodies.
As described herein, the detection of anti-L IgA and anti-C IgA is used as a prognostic tool for CD patients.
As described herein, anti-L and anti-C detection is used to predict the course of CD and is a very important aid for clinicians in determining optimal patient treatment. A positive (determined by cutoff values or reference level) anti-L assay, anti-C assay, or combined anti-L and anti-C assay is associated with a more complicated disease behavior (fistulas or stenosis) and the need for abdominal surgery, while a negative anti-L assay, a negative anti-C assay, or a negative combined anti-L and anti-C assay is associated with the mild inflammatory disease phenotype. Moreover, higher levels of anti-L and anti-C antibodies are also associated with a more complicated disease behavior and the need for abdominal surgery. By contrast, lower levels of anti-L or anti-C antibodies are associated with the mild inflammatory CD phenotype. The antibodies identified by IBDX® are also associated with CD phenotype.
Notably, the sera antibody reactivity to chitosan lactate is different from the reactivity to chitobioside. Additionally, the sera antibody reactivity to laminarin is different from the reactivity to laminaribioside.
The detection of high levels of anti-L, anti-C, alone or in combination with gASCA, enables the identification of CD patients who may require early surgery and/or who are susceptible to develop complications, and may need to undergo surgery early and repeatedly during the course of the disease. These patients should be targeted for more aggressive therapeutic management.
The clinical parameters of sensitivity, specificity, negative predictive value, positive predictive value and efficiency are calculated using true positives, false positives, false negatives and true negatives. A “true positive” sample is a sample positive for CD according to art-recognized methods for diagnosing/prognosing CD, which is also diagnosed positive according to a method of the invention. A “false positive” sample is a sample negative by an art-recognized method, which is diagnosed positive according to a method of the invention. Similarly, a “false negative” is a sample positive for an art-recognized analysis, which is diagnosed negative according to a method of the invention. A “true negative” is a sample negative for the assessed trait by an art-recognized method, and also negative 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 the measured trait. 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 antibody values can be selected such that the sensitivity of diagnosing/prognosing an individual is at least about 20%, and can be, for example, at least about 30%, 40%, 50%, 60%, 70%, 80% or 90%.
As used herein, the term “specificity” means the probability that a method is negative in the absence of the measured trait. 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 the measured trait. The cut-off value can be selected such that, when the sensitivity is at least about 30%, the specificity of diagnosing/prognosing an individual is in the range of 80-99%, for example, 80-85%, 85-90%, 90-95% or 95-99%.
The study population included 1144 clinically well-defined, frozen sera samples from CD patients and controls. Samples taken from patients fulfilling the following requirements were included in the study: Age 18-60 years at time of sampling and definite diagnosis as CD or non CD controls. The present analysis included: (1) Crohn's Disease patients—549 (52% female, mean age±SD, 33.4±13.9 years); and (2) Non-CD controls—595 mean age (SD), (53% female, mean age±SD, 40.7±15.5 years) comprised of: (i) Ulcerative Colitis patients—333; (ii) Irritable Bowel Disease patients—30; (iii) Indeterminate Colitis patients-36; (iv) Healthy subjects—94; and (v) Unaffected CD relatives—102.
For the detection of anti-L antibodies, Nunc plates (Cat. no. 445101, Denmark) were activated with cyanuric chloride (Cat. No., SI-C9550-1, Sigma-Aldrich, St Louis, Mo., USA) and washed with deionized water. Immediately after washing, wells were filled with 100 μl Laminarin (Cat. No., 109-035-011, Sigma-Aldrich, St Louis, Mo., USA), diluted in phosphate-buffered saline (PBS), pH 7.4 (Cat. No. P-3813, Sigma-Aldrich, St Louis, Mo., USA) to a final concentration of 100 μg/ml. After overnight incubation at room temperature (RT), plates were washed with deionized water and incubated 15 min with methanol (Cat. No. 830108213, Gadot, Israel) and dried at RT.
For the detection of anti-C antibodies, wells were filled with 100 μl chitosan lactate (Cat. No. 523682-1G, Sigma-Aldrich, St Louis, Mo., USA) and diluted in PBS to a final concentration of 200 μg/ml. After overnight incubation at RT, plates were washed with deionized water and dried at RT.
Patient's sera were thawed and diluted at 1:101 in IBDX™ Sample Diluent (Cat. No. G300023, Glycominds Ltd., Lod, Israel) one day before assay and kept at 4° C. until addition to plates. Diluted sera were dispensed 100 μl/well and incubated at RT for 30 min. Plates were washed with Wash Solution (Cat. No. G300022, Glycominds, Lod, Israel) and incubated for 30 min with 100 μl peroxidase conjugated AffiniPure goat anti human sera IgA, a chain specific (Cat. No. 109-035-011, Jackson Immunoresearch, PA, USA) diluted 1:12.500 for Anti-L detection and 1:4000 for Anti-C detection in 50% Stabilizyme HRP (Cat. No. SZ02-2000, SurModics, MN, USA) 50% Water (Cat. No. 03-055-1A, Biological Industries, Israel) to a final concentration of 32 ng/ml for anti-L detection and 0.1 ng/ml for anti-C detection. After 30 minutes incubation at RT, unbound antibodies were removed by washing with Wash Solution. TMB chromogen (Cat. No. RM 1928, Savyon, Israel) was added for 30 minutes and HRP-Stop solution (Cat. No. G300021, Glycominds, Lod, Israel) was added to terminate the enzymatic reaction and absorbance was measured with Victor spectrophotometer (Wallac, Turku, Finland) at 450 nm. A reference sample for each antibody was used to calibrate the antibody levels. Results are reported in arbitrary EIA units (EU). Samples were identified as positive for a particular antibody if the antibody levels were above a reference level. The reference levels for anti-C (90 units) and anti-L (60 units) were set as the upper 95% of the non-CD population.
Detection of Antibodies with IBDX™
The detection of gASCA, ALCA, ACCA and AMCA with the capture reagents in IBDX® was performed according to the manufacturer's instructions (Cat. no. S701100, L703100, C702100 and M704100, Glycominds, Lod, Israel). Results are reported in arbitrary ELISA units (EU). Patients were identified as positive for a particular antibody based on cut-off levels, which were provided by the manufacturer for differentiation between CD and non CD patients.
The level of anti-C and anti-L was compared between CD to non-CD populations by Mann-Whitney U and box-plots. The association of antibody positivity to clinical phenotype was performed by χ2 test for nominal data and Mann-Whitney U for continuous data. The association of antibody level to clinical phenotype was performed by Mann-Whitney U. p<0.05 was considered significant. The correlation between anti-polysaccharides and their fragment was calculated using Pearson methods and reported as R2.
For the CD population two by two tables were created (for χ2 analyses) according to the following criteria: antibody positivity [positive/negative anti-L, positive/negative anti-C, positive/negative anti-L and/or anti-C (anti-L/C)]; and disease phenotypes (yes or no) for: ileal location (L1), colon location (L2), inflammatory disease behavior (B1), stricturing disease behavior (B2), penetrating disease behavior (B3), perianal disease behavior (P), and if the patient had a prior abdominal surgery. In addition, the disease duration, age of diagnosis and number of surgeries were compared according to antibody positivity (positive/negative anti-L, positive/negative anti-C, combined positive/negative anti-L and/or anti-C). Results for antibody positivity, as described below, are shown in Table 1.
A significantly higher proportion of CD patients positive for anti-L had penetrating (B3) and perianal (P) disease behavior, and prior abdominal surgery compared to patients negative for anti-L. Also, a significantly higher proportion of CD patients having the mild inflammatory (B1) disease behavior were negative for anti-L compared to patients positive for anti-L. CD patients positive for anti-L also had a longer disease duration and higher number of abdominal surgeries than CD patients negative for anti-L. No association was found between anti-L positivity to age of diagnosis, ileal location (L1), colon location (L2) or to stricturing (B2) phenotypes.
A significantly higher proportion of CD patients that were positive for anti-C had penetrating (B3) and perianal (P) disease behavior and prior abdominal surgery compared to patients negative for anti-C. CD patients positive for anti-C had a lower age of diagnosis and higher number of abdominal surgeries than patients negative for anti-C. No association was found between anti-C positivity to disease duration, ileal location (L1), colon location (L2) or stricturing (B2) disease behavior.
A significantly higher proportion of CD patients positive for the combined anti-L and/or anti-C analysis had penetrating (B3) and perianal (P) disease behavior and prior abdominal surgery than patients negative for the combined analysis. Also, a significantly higher proportion of CD patients negative for the combined anti-L and/or anti-C analysis had the inflammatory (B1) disease behavior. CD patients positive for the combined analysis had a higher number of abdominal surgeries and longer disease duration than patients negative for the combined analysis. No association was found between the combined anti-L and/or anti-C positivity to ileal location (L1), colon location (L2) or to stricturing (B2) disease behavior.
Table 2 shows the level of anti-C and anti-L in various CD phenotypes (yes or no) for: inflammatory (B1) disease behavior, stricturing (B2) disease behavior, penetrating (B3) disease behavior, perianal (P) disease behavior and prior abdominal surgery.
Patients with prior abdominal surgery had higher levels of anti-L than patients without prior abdominal surgery. The level of anti-L was also higher among patients with penetrating (B3) and perianal (P) disease behavior compared to patients without stricturing (B2), penetrating (B3) or perianal (P) disease behavior. Patients with the mild inflammatory disease behavior (B1) had lower levels of Anti-L than patients without the B1 disease behavior. The level of Anti-L was not associated with stricturing disease (B2) behavior.
Patients with prior abdominal surgery had higher levels of anti-C than patients without prior abdominal surgery. The level of anti-C was also higher amongst patients with stricturing (B2), penetrating (B3) and perianal (P) disease behavior than patients without stricturing (B2), penetrating (B3) or perianal (P) diseases behavior. Patients with the mild inflammatory disease behavior (B1) had lower levels of anti-C than patients without the B1 disease behavior.
Patients with prior abdominal surgery had higher levels of gASCA than patients without prior abdominal surgery. The level of gASCA was also higher among patients with stricturing (B2) and penetrating (B3) disease behavior compared to patients without stricturing (B2) or penetrating (B3) diseases behavior. Patients with the mild inflammatory disease behavior (B1) had lower levels of gASCA than patients without the B1 disease behavior. The level of gASCA was not associated with perianal disease behavior (P).
Patients with prior abdominal surgery had higher levels of ALCA than patients without prior abdominal surgery. Patients with the mild inflammatory disease behavior (B1) had lower levels of ALCA than patients without the B1 disease behavior. The level of ALCA was not associated with stricturing disease behavior (B2), penetrating disease behavior (B3) or perianal disease behavior (P).
Patients with prior abdominal surgery had higher levels of ACCA than patients without prior abdominal surgery. The level of ACCA was also higher amongst patients with penetrating (B3) or perianal (P) disease behavior compared to patients without B3, or P diseases behavior. Patients with the mild inflammatory disease behavior (B1) had lower levels of ACCA than patients without the B1 disease behavior. The level of ACCA was not associated with stricturing disease behavior (B2).
Patients with prior abdominal surgery had higher levels of AMCA than patients without prior abdominal surgery. The level of AMCA was also higher amongst patients with penetrating (B3) or perianal (P) disease behavior compared to patients without B3, or P diseases behavior. Patients with the mild inflammatory disease behavior (B1) had lower levels of AMCA than patients without the B1 disease behavior. The level of AMCA was not associated with stricturing disease behavior (B2).
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Laminarin (also known as laminaran) is a storage glucan (a polysaccharide of glucose) of Laminaria and other brown algae (
These results demonstrate a lack of correlation (R2 0.15-0.17) between anti-glycan antibodies reactive to polysaccharides or fragments of the polysaccharides. In both cases there were patients who were highly reactive towards the polysaccharide, but were not reactive towards the defined sub-fragment and vice versa, demonstrating the unpredictable nature of the association. Therefore, one cannot assume that if an individual has developed an immune response to a polysaccharide, he will also react immunologically to a sub-fragment of the polysaccharide.
Crohn's disease (CD) often leads to complications like fistulae and stenoses, making a surgical intervention necessary. A panel of serological antibodies including the previously unpublished Anti-Laminarin IgA (Anti-L) and Anti-Chitin IgA antibodies were examined for their accuracy in predicting the early appearance of complicated disease or surgery in CD.
Serum samples of 142 CD patients (mean age 33.7 years, 42% female, mean disease duration 52.6 months, procurement 2000-2005) and a control group including samples from 124 ulcerative colitis (UC), 74 other gastrointestinal diseases (OGD) and 249 healthy controls (HC) were tested for the presence of Anti-L, Anti-C, Anti-Chitobioside IgA Carbohydrate Antibodies (ACCA), Anti-Laminaribioside Carbohydrate IgG Antibodies (ALCA), Anti-Mannobioside Carbohydrate IgG Antibodies (AMCA) and Anti Saccaromyces cervisiae IgG Antibodies (gASCA) by ELISA (Glycominds Ltd., Lod, Israel). No CD patient had fistulas, stenoses or abdominal surgery before or at the time of sample procurement. CD patients were followed up regularly and clinical data were reviewed for the appearance of a complicated disease course or CD related surgery.
For the detection of anti-L antibodies, Nunc plates (Cat. no. 445101, Denmark) were activated with cyanuric chloride (Cat. No., SI-C9550-1, Sigma-Aldrich, St Louis, Mo., USA) and washed with deionized water. Immediately after washing, wells were filled with 100 μl Laminarin (Cat. No., 109-035-011, Sigma-Aldrich, St Louis, Mo., USA) diluted in phosphate-buffered saline (PBS), pH 7.4 (Cat. No. P-3813, Sigma-Aldrich, St Louis, Mo., USA) to a final concentration of 100 μg/ml. After overnight incubation at room temperature (RT), plates were washed with deionized water and incubated 15 min with methanol (Cat. No. 830108213, Gadot, Israel) and dried at RT.
For the detection of anti-C antibodies, wells were filled with 100 μl chitosan lactate (Cat. No. 523682-1G, Sigma-Aldrich, St Louis, Mo., USA) diluted in PBS to a final concentration of 200 μg/ml. After overnight incubation at RT, plates were washed with deionized water and dried at RT.
Patient's sera were thawed and diluted at 1:101 in IBDX™ Sample Diluent (Cat. No. G300023, Glycominds Ltd., Lod, Israel) one day before assay and kept at 4° C. until addition to plates. Diluted sera were dispensed 100 μl/well and incubated at RT for 30 min. Plates were washed with Wash Solution (Cat. No. G300022, Glycominds, Lod, Israel) and incubated for 30 min with 100 μl peroxidase conjugated AffiniPure goat anti human sera IgA, α chain specific (Cat. No. 109-035-011, Jackson Immunoresearch, PA, USA) diluted 1:12.500 for Anti-L detection and 1:4000 for Anti-C detection in 50% Stabilizyme HRP (Cat. No. SZ02-2000, SurModics, MN, USA) 50% Water (Cat. No. 03-055-1A, Biological Industries, Israel) to final concentration of 32 ng/ml for anti-L detection and 0.1 ng/ml for anti-C detection. After 30 minutes incubation at RT, unbound antibodies were removed by washing with Wash Solution. TMB chromogen (Cat. No. RM 1928, Savyon, Israel) was added for 30 minutes and HRP-Stop solution (Cat. No. G300021, Glycominds, Lod, Israel) was added to terminate the enzymatic reaction and absorbance was measured with Victor spectrophotometer (Wallac, Turku, Finland) at 450 nm. A reference sample for each antibody was used to calibrate the antibody levels. Results are reported in arbitrary EIA units (EU).
Patients sample were considered positive for anti-C or anti-L if the levels were above a certain cutoff EU value. The cutoff value for determination of positivity for anti-C or anti-L was set based on the upper 95% percentile of a non CD control population.
Detection of Antibodies with IBDX™
The detection of gASCA, ALCA, ACCA and AMCA with capture reagents in IBDX® was performed according to the manufacturer's instructions (Cat. no. S701100, L703100, C702100 and M704100, Glycominds, Lod, Israel). Results are reported in arbitrary ELISA units (EU). Patients were determined as positive for the antibody if levels were above cutoff value define by the manufacturer
Kaplan-Meier estimates, together with log rank tests, Hazard Ratio (HR), and Cox proportional hazard regression models, were used to assess the association between antibodies level (EU and positive status—below or above cutoff) and risk for the development of fistulas or stenosis or having an abdominal surgery.
Median follow-up was 55.4 months (min. 18.2, max. 78.3). 48.6% of the patients developed fistulae or stenoses and 25.3% of the patients had abdominal surgery during follow up. Kaplan-Meier estimates, together with log rank tests and Cox proportional hazard regression models, showed that patients positive for certain anti-glycan antibodies had a significantly higher probability for the appearance of fistulas or stenoses (ACCA, p=0.0072, hazard ratio (HR) 2.0; AMCA p=0.0005, HR 2.4; gASCA p=0.0045, HR 1.9; Anti-L p=0.0002, HR 2.6). In addition, patients positive for AMCA (p=0.045, HR 2.0) or Anti-L (p=0.0007 HR 3.4) had a higher probability of abdominal surgery. CD patients positive for two or more markers had an higher risk for the development of fistulas or stenoses (p=0.005 HR 2.4) and this was also found to be an independent prognostic variable in a Cox regression model corrected for BMI, age, sex, disease duration and small bowel disease location (p=0.008, HR 2.2). CD patients positive for two or more of the antibodies Anti-L, Anti-C, AMCA or gASCA had a higher probability for early surgery (p=0.008, HR 2.8). Moreover, in the Cox regression model this was the only present independent prognostic variable (p<0.001, HR 3.1). The data indicate that the novel anti-glycan antibodies, Anti-L and Anti-C, in conjunction with ALCA, ACCA, gASCA and AMCA, can prospectively predict early development of fistulas or stenosis and surgery in patients with Crohn's disease.
The results of the above-identified experiments are depicted in
For example,
This application claims priority to U.S. Ser. No. 61/005,060, filed Nov. 30, 2007, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/003927 | 11/26/2008 | WO | 00 | 8/10/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61005060 | Nov 2007 | US |
