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• W2030421888 abstract "Background & Aims: New serologic markers of inflammatory bowel disease may be useful for differentiating between Crohn’s disease and ulcerative colitis and for disease stratification. We profiled sugar-binding antibodies to identify novel antiglycan antibodies that may be associated with inflammatory bowel disease. Methods: Serum samples were obtained from patients with diagnosed Crohn’s disease or ulcerative colitis and from control patients. The presence of antiglycan antibodies was evaluated using either a glycan array (GlycoChip; Glycominds, Ltd, Lod, Israel) in patients with Crohn’s disease (n = 72) or ulcerative colitis (n = 56) and in healthy controls (n = 41) or using an enzyme-linked immunosorbent assay in patients with Crohn’s disease (n = 124), ulcerative colitis (n = 106), and in control patients (n = 101). Results: Inaddition to antibodies against mannan, antibodies to laminaribioside (Glc[β1,3]Glc[β]) and chitobioside (GlcNAc[β1,4]GlcNAc[β]) had the highest discriminative capability between Crohn’s disease and ulcerative colitis (P < .001 and P < .05, respectively). Importantly, 44% (12/27) of anti-Saccharomyces cerevisiae antibody-negative Crohn’s disease patients were positive for antilaminaribioside or antichitobioside. In patients with inflammatory bowel disease positive for antibodies against either laminaribioside, chitobioside, or mannan, the diagnosis of Crohn’s disease was suggested with a sensitivity of 77.4% and specificity of 90.6%. Having at least 2 of these antibodies increased the specificity to 99.1%. In Crohn’s disease, higher levels of antibodies against laminaribioside or mannan were significantly associated with small intestinal disease (P = .03 and P < .0001, respectively). Conclusions: Antilaminaribioside and antichitobioside carbohydrate antibodies are novel serologic markers associated with Crohn’s disease. These antibodies may contribute to the diagnosis and improved stratification of Crohn’s disease. Background & Aims: New serologic markers of inflammatory bowel disease may be useful for differentiating between Crohn’s disease and ulcerative colitis and for disease stratification. We profiled sugar-binding antibodies to identify novel antiglycan antibodies that may be associated with inflammatory bowel disease. Methods: Serum samples were obtained from patients with diagnosed Crohn’s disease or ulcerative colitis and from control patients. The presence of antiglycan antibodies was evaluated using either a glycan array (GlycoChip; Glycominds, Ltd, Lod, Israel) in patients with Crohn’s disease (n = 72) or ulcerative colitis (n = 56) and in healthy controls (n = 41) or using an enzyme-linked immunosorbent assay in patients with Crohn’s disease (n = 124), ulcerative colitis (n = 106), and in control patients (n = 101). Results: Inaddition to antibodies against mannan, antibodies to laminaribioside (Glc[β1,3]Glc[β]) and chitobioside (GlcNAc[β1,4]GlcNAc[β]) had the highest discriminative capability between Crohn’s disease and ulcerative colitis (P < .001 and P < .05, respectively). Importantly, 44% (12/27) of anti-Saccharomyces cerevisiae antibody-negative Crohn’s disease patients were positive for antilaminaribioside or antichitobioside. In patients with inflammatory bowel disease positive for antibodies against either laminaribioside, chitobioside, or mannan, the diagnosis of Crohn’s disease was suggested with a sensitivity of 77.4% and specificity of 90.6%. Having at least 2 of these antibodies increased the specificity to 99.1%. In Crohn’s disease, higher levels of antibodies against laminaribioside or mannan were significantly associated with small intestinal disease (P = .03 and P < .0001, respectively). Conclusions: Antilaminaribioside and antichitobioside carbohydrate antibodies are novel serologic markers associated with Crohn’s disease. These antibodies may contribute to the diagnosis and improved stratification of Crohn’s disease. See editorial on page 664. See editorial on page 664. Inflammatory bowel disease (IBD) is a chronic intestinal disorder of unknown etiology comprising 2 major types: Crohn’s disease (CD) and ulcerative colitis (UC). The diagnosis of IBD and the differentiation between UC and CD are established by the combination of clinical, laboratory, radiologic, endoscopic, histopathologic, and serologic characteristics. However, when a definite diagnosis cannot be established, as is true in 10%–17% of colitis patients, a diagnosis of indeterminate colitis (IC) is designated.1Hodgson H.J.F. Ulcerative colitis versus Crohn’s disease-one disease or two?.in: Allan R.N. Rhodes J.M. Hanauer S.B. Keighley M.R.B. Alexander-Williams J. Fazio V.W. Inflammatory bowel diseases. 3rd ed. Churchill Livingstone, New York1997: 343-347Google Scholar, 2Joossens S. Reinisch W. Vermeire S. Sendid B. Poulain D. Peeters M. Geboes K. Bossuyt X. Vandewalle P. Oberhuber G. Vogelsang H. Rutgeerts P. Colombel J.F. The value of serologic markers in indeterminate colitis a prospective follow-up study.Gastroenterology. 2002; 122: 1242-1247Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 3Moum B. Ekbom A. Vatn M.H. Aadland E. Sauar J. Lygren I. Schulz T. Stray N. Fausa O. Inflammatory bowel disease re-evaluation of the diagnosis in a prospective population based study in south eastern Norway.Gut. 1997; 40: 328-332PubMed Google Scholar, 4Price A.B. Overlap in the spectrum of nonspecific inflammatory bowel disease—“colitis indeterminate.”.J Clin Pathol. 1978; 31: 567-577Crossref PubMed Scopus (316) Google Scholar, 5Riegler G. Arimoli A. Esposito P. Iorio R. Carratu R. Clinical evolution in an outpatient series with indeterminate colitis.Dis Colon Rectum. 1997; 40: 437-439Crossref PubMed Scopus (20) Google Scholar In addition to diagnosing IBD, serologic markers may be used to discriminate between CD and UC, assess disease activity and progression, predict response to treatment, and stratify disease severity.6Targan S. Serology and laboratory markers of disease activity.in: Sartor R.B. Sandborn W.J. Kirsner’s inflammatory bowel disease. 6th ed. Saunders, New York2004: 442-452Google Scholar The major serologic markers for IBD in commercial use are antineutrophil cytoplasmic antibodies (ANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA). ANCA are autoantibodies directed at antigens found mostly in azurophilic granules of neutrophils7Reumaux D. Sendid B. Poulain D. Duthilleul P. Dewit O. Colombel J.F. Serological markers in inflammatory bowel diseases.Best Pract Res Clin Gastroenterol. 2003; 17: 19-35Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 8Saxon A. Shanahan F. Landers C. Ganz T. Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease.J Allergy Clin Immunol. 1990; 86: 202-210Abstract Full Text PDF PubMed Scopus (496) Google Scholar and have a prevalence of up to 85% among UC patients and 5%–28% among CD patients and healthy people. ASCA are directed against mannose residues on mannan in the cell walls of the yeast S cerevisiae9McKenzie H. Main J. Pennington C.R. Parratt D. Antibody to selected strains of Saccharomyces cerevisiae (baker’s and brewer’s yeast) and Candida albicans in Crohn’s disease.Gut. 1990; 31: 536-538Crossref PubMed Scopus (145) Google Scholar, 10Sendid B. Colombel J.F. Jacquinot P.M. Faille C. Fruit J. Cortot A. Lucidarme D. Camus D. Poulain D. Specific antibody response to oligomannosidic epitopes in Crohn’s disease.Clin Diagn Lab Immunol. 1996; 3: 219-226PubMed Google Scholar and have a prevalence of 48%–69% among CD patients and 15% among UC patients.7Reumaux D. Sendid B. Poulain D. Duthilleul P. Dewit O. Colombel J.F. Serological markers in inflammatory bowel diseases.Best Pract Res Clin Gastroenterol. 2003; 17: 19-35Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 10Sendid B. Colombel J.F. Jacquinot P.M. Faille C. Fruit J. Cortot A. Lucidarme D. Camus D. Poulain D. Specific antibody response to oligomannosidic epitopes in Crohn’s disease.Clin Diagn Lab Immunol. 1996; 3: 219-226PubMed Google Scholar It has been demonstrated that serologic markers are associated with phenotypic subsets of CD. ASCA is associated with severe small bowel disease rather than colonic disease and with penetrating CD, whereas ANCA-positive CD patients have a more “UC-like” clinical manifestation.11Vasiliauskas E.A. Kam L.Y. Karp L.C. Gaiennie J. Yang H. Targan S.R. Marker antibody expression stratifies Crohn’s disease into immunologically homogeneous subgroups with distinct clinical characteristics.Gut. 2000; 47: 487-496Crossref PubMed Scopus (294) Google Scholar, 12Vasiliauskas E.A. Plevy S.E. Landers C.J. Binder S.W. Ferguson D.M. Yang H. Rotter J.I. Vidrich A. Targan S.R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn’s disease define a clinical subgroup.Gastroenterology. 1996; 110: 1810-1819Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Walker L.J. Aldhous M.C. Drummond H.E. Smith B.R. Nimmo E.R. Arnott I.D. Satsangi J. Anti-Saccharomyces cerevisiae antibodies (ASCA) in Crohn’s disease are associated with disease severity but not NOD2/CARD15 mutations.Clin Exp Immunol. 2004; 135: 490-496Crossref PubMed Scopus (90) Google Scholar Other antibody markers have been reported in serum samples from IBD patients, including those against outer membrane porin C (OmpC) of Escherichia coli, found in 38%–55% of CD and 2% of UC patients, against the bacterial sequence I2, found in 54% of CD and 10% of UC patients, and against bacterial flagellin (CBir), in CD, in contrast to UC patients.14Cohavy O. Bruckner D. Gordon L.K. Misra R. Wei B. Eggena M.E. Targan S.R. Braun J. Colonic bacteria express an ulcerative colitis pANCA-related protein epitope.Infect Immun. 2000; 68: 1542-1548Crossref PubMed Scopus (174) Google Scholar, 15Sutton C.L. Kim J. Yamane A. Dalwadi H. Wei B. Landers C. Targan S.R. Braun J. Identification of a novel bacterial sequence associated with Crohn’s disease.Gastroenterology. 2000; 119: 23-31Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Lodes M.J. Cong Y. Elson C.O. Mohamath R. Landers C.J. Targan S.R. Fort M. Hershberg R.M. Bacterial flagellin is a dominant antigen in Crohn disease.J Clin Invest. 2004; 113: 1296-1306Crossref PubMed Scopus (675) Google Scholar, 17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar These antibodies, directed at bacterial antigens, are also associated with distinct disease phenotypes, specifically more complicated small bowel disease.17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 18Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar However, the significance of serologic markers for discrimination between UC and CD, and their significance in better classifying IC, remains controversial.2Joossens S. Reinisch W. Vermeire S. Sendid B. Poulain D. Peeters M. Geboes K. Bossuyt X. Vandewalle P. Oberhuber G. Vogelsang H. Rutgeerts P. Colombel J.F. The value of serologic markers in indeterminate colitis a prospective follow-up study.Gastroenterology. 2002; 122: 1242-1247Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 19Quinton J.F. Sendid B. Reumaux D. Duthilleul P. Cortot A. Grandbastien B. Charrier G. Targan S.R. Colombel J.F. Poulain D. Anti-Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease prevalence and diagnostic role.Gut. 1998; 42: 788-791Crossref PubMed Scopus (541) Google Scholar, 20Ruemmele F.M. Targan S.R. Levy G. Dubinsky M. Braun J. Seidman E.G. Diagnostic accuracy of serologic assays in pediatric inflammatory bowel disease.Gastroenterology. 1998; 115: 822-829Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar Therefore, there remains a considerable unmet need for serologic markers in IBD. The existence of antibodies against sugars, such as ASCA, that are CD specific and the need for better serologic stratification of IBD prompted us to perform an extensive antiglycan antibody search in IBD patients. Glycans are predominant surface components of cells such as erythrocytes, immune cells, and microorganisms. As such, they give rise to high levels of antiglycan antibodies of all classes, which have been demonstrated in a number of inflammatory and autoimmune diseases.21Buonomano R. Tinguely C. Rieben R. Mohacsi P.J. Nydegger U.E. Quantitation and characterization of anti-Galα1-3Gal antibodies in sera of 200 healthy persons.Xenotransplantation. 1999; 6: 173-180Crossref PubMed Scopus (63) Google Scholar, 22McMorrow I.M. Comrack C.A. Sachs D.H. DerSimonian H. Heterogeneity of human anti-pig natural antibodies cross-reactive with the Gal(α1,3)Galactose epitope.Transplantation. 1997; 64: 501-510Crossref PubMed Scopus (102) Google Scholar, 23Willison H.J. Yuki N. Peripheral neuropathies and anti-glycolipid antibodies.Brain. 2002; 125: 2591-2625Crossref PubMed Scopus (612) Google Scholar To investigate antiglycan antibodies as potential serologic markers in IBD, we first used an array of sugars (mono-, di-, and trisaccharides) to characterize the antiglycan antibody profile in serum samples from CD and UC patients and from healthy controls (the discovery phase). We then screened serum samples from IBD patients and controls to detect the presence of the characteristic antiglycan antibodies (the validation phase). Here, we report on the discovery of 2 novel antiglycan antibodies, directed against laminaribioside and chitobioside, that appear to be associated with CD. Patients (N = 331) were recruited at the Department of Gastroenterology and Liver Diseases, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. Signed informed consent forms were obtained from all patients, and the local ethical committee approved the study. The diagnosis of UC or CD was based on established clinical, radiographic, endoscopic, and histopathologic criteria. Patient characteristics were determined from medical records, questionnaires, and interviews. Ashkenazi or Sephardic origins were assigned on the basis of the birthplace of the 4 grandparents. Mixed origin is the case for which at least 1 grandparent belonged to a different ethnic Jewish group than the other grandparents. Patients were considered current or past smokers if they smoked a minimum of 7 cigarettes per week for at least 1 year anytime during their life. CD patients were assigned phenotypes based on previously published criteria.24Gasche C. Scholmerich J. Brynskov J. D’Haens G. Hanauer S.B. Irvine E.J. Jewell D.P. Rachmilewitz D. Sachar D.B. Sandborn W.J. Sutherland L.R. A simple classification of Crohn’s disease report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998.Inflamm Bowel Dis. 2000; 6: 8-15Crossref PubMed Scopus (1052) Google Scholar Disease behavior was divided based on the Vienna classification into nonstricturing nonpenetrating (inflammatory), stricturing, and penetrating disease types. Disease location was defined as the presence of inflammation in the small bowel, colon, or both on the basis of endoscopic, radiologic, or histopathologic studies. Phenotypes and disease locations were assigned by IBD physicians once the disease was already established. The control groups consisted of healthy blood donors (HC), and, for the validation screening (see in “Serologic Analysis” section), we also added a non-IBD gastrointestinal diseases control group (OGD, n = 61). This group consisted of celiac disease patients (n = 27) confirmed by serology (antibodies to tissue transglutaminase and to endomysium) as well as by duodenal biopsies; irritable bowel syndrome (IBS) patients (n = 20); and 14 patients with colonic polyps, diverticular disease, pseudomembranous colitis, Helicobacter pylori gastritis, pancreatitis, and hemorrhoids. Patient characteristics are presented in Table 1, Table 2.Table 1Patient Characteristics in the Discovery ScreeningCD (n = 72)UC (n = 56)HC (n = 41)Age, y (range)35.5 (19–70)40.9 (14–89)38.9 (18–78)Female, n (%)31 (43)28 (50)21 (51)Mean disease duration, y (range)9.1 (1–54)7.9 (1–39)—Mean age at diagnosis, y (range)26.6 (8–63)33 (10–87)—Bowel surgery (including appendectomy), n (%)28 (38.9)3 (5.4)aP < .0001 vs CD.—Extraintestinal manifestations, n (%)28 (38.9)14 (25.0)—Treatment: 5-ASA, n (%)48 (66.7)53 (94.6)aP < .0001 vs CD.— Antibiotics, n (%)15 (20.8)4 (7.1)— Steroids, n (%)23 (31.9)15 (26.7)— 6MP/AZA, n (%)22 (30.5)12 (21.4)— MTX, n (%)5 (6.9)0 (0)— Infliximab, n (%)9 (12.5)0 (0)bP < .05 vs CD.— No treatment, n (%)0 (0)0 (0)—6-MP, 6-mercaptopurine; AZA, azathioprine; MTX, methotrexate.a P < .0001 vs CD.b P < .05 vs CD. Open table in a new tab Table 2Patient Characteristics in the Validation ScreeningCD (n = 124)UC (n = 106)OGD (n = 61)HC (n = 40)Mean age, y (range)35.3 (16–71)41.3 (14–89)aP < .0001 vs CD.33.7 (1–83)37.0 (18–78)Female, n (%)48 (39)49 (46)31 (50)21 (51)IBD patient characteristics Mean disease duration, y (range)8.1 (1–54)8.0 (1–39) Mean age at diagnosis, y (range)26.9 (8–65)32.1 (10–87) Bowel surgery (including appendectomy), n (%)41 (33)3 (2)aP < .0001 vs CD. Extra intestinal manifestations, n (%)47 (37)22 (20) Smoking status: Current and past smoker, n (%)57 (46)36 (34) Nonsmoker, n (%)66 (53)59 (56) Unknown, n (%)1 (1)11 (10) Origin Ashkenazi, n (%)65 (52)52 (49) Sephardic, n (%)46 (37)23 (22) Mixed, n (%)12 (10)11 (10) Non-Jewish, n (%)0 (0)8 (8) Unknown, n (%)1 (1)12 (11)CD characteristics Disease location Small bowel, n (%)67 (54)— Colon, n (%)17 (14)— Both, n (%)40 (32)— Disease behaviorsbDisease behavior was classified based on the Vienna classification.24 Inflammatory, n (%)57 (46)— Stricturing, n (%)38 (31)— Penetrating, n (%)29 (23)— Mean CDAI (SD)185 (121)—NOTE. OGD group included 27 celiac disease patients; 20 irritable bowel syndrome (IBS) patients; and 14 patients with colonic polyps, diverticular disease, pseudomembranous colitis, Helicobacter pylori gastritis, pancreatitis, and hemorrhoids.CDAI, Crohn’s disease activity index.a P < .0001 vs CD.b Disease behavior was classified based on the Vienna classification.24Gasche C. Scholmerich J. Brynskov J. D’Haens G. Hanauer S.B. Irvine E.J. Jewell D.P. Rachmilewitz D. Sachar D.B. Sandborn W.J. Sutherland L.R. A simple classification of Crohn’s disease report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998.Inflamm Bowel Dis. 2000; 6: 8-15Crossref PubMed Scopus (1052) Google Scholar Open table in a new tab 6-MP, 6-mercaptopurine; AZA, azathioprine; MTX, methotrexate. NOTE. OGD group included 27 celiac disease patients; 20 irritable bowel syndrome (IBS) patients; and 14 patients with colonic polyps, diverticular disease, pseudomembranous colitis, Helicobacter pylori gastritis, pancreatitis, and hemorrhoids. CDAI, Crohn’s disease activity index. Blood samples were collected in evacuated silicon-coated tubes containing gel for easier separation between serum and blood clot (Cat. 418XX3GLV; Estar Technologies, Holon, Israel). After blood coagulation, serum was separated by centrifugation at 100g, collected, and kept frozen at −80°C until use. The laboratory evaluations were conducted in a blinded manner. Profiling of antiglycan antibodies in serum samples was done using GlycoChip (Glycominds, Ltd, Lod, Israel), a glycan array in the 384-well microtiter plate format, as previously described.25Schwarz M. Spector L. Gargir A. Shtevi A. Gortler M. Altstock R.T. Dukler A.A. Dotan N. A new kind of carbohydrate array, its use for profiling antiglycan antibodies, and the discovery of a novel human cellulose-binding antibody.Glycobiology. 2003; 13: 749-754Crossref PubMed Scopus (127) Google Scholar Briefly, p-nitrophenyl saccharides were covalently bound to the surface of the microtiter plate wells (384-well reduced-volume black microtiter plate; Greiner Bio-One International AG, Kremsmuenster, Austria) with a linker (an oligomer of 1,8-diamino-3,6-dioxaoctan [DD8]; Sigma Chemical Co., St. Louis, MO). The mono-, di-, oligo-, and polysaccharides used are listed in Table 3.Table 3Glycans Displayed on the Glycochip Glycan ArrayGlycan numberIUPAC nomenclatureGlycan numberIUPAC nomenclature1Gal(β) 3-sulfate25GlcNAc(β1,4)GlcNAc(β)2Gal(α)26GlcNAc(β1,3)Gal(β1,4)Glc(β)3Gal(β)27GlcNAc(β1,6)GalNAc(α)4Gal(β1,3)[GlcNAc(β1,6)]GalNAc(α)28L-Rha(α)5Gal(β1,3)GalNAc(α)29GalA(β)6Gal(β1,3)GlcNAc(β)30Man(α)7Gal(β1,4)Glc(β)31Man(α1,3)Man(α)8Gal(β1,4)GlcNAc(α)32Man(β)9Gal(β1,3)Gal(β1,4)GalNAc(β)33Man(β1,4)Glc(β)10Gal(β1,6)Gal(β)34Neu5Ac(α)11GalNAc(α)35L-Ara(α)12GalNAc(β)36GlcA(β)13Fuc(α)37Xyl(α)14Fuc(α1,2)Gal(β)38Xyl(β)15Fuc(β)39Gal(α1,3)Gal(β1,4)GlcNAc(β)16Glc(α)40Gal(α1,3)Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc(β)17Glc(α1,4)Glc(α)41Gal(α1,4)Gal(β1,4)Glc(β)18Glc(α1,4)Glc(β)42Glc(β1,4)Glc(β)19Glc(β)43Man(α1,2)Manα20Glc(β1,3)Glc(β)44Man(α1,3)[Man(α1,6)]Man(β)21GlcNAc(β) 6-sulfate46Man(α1,6)Manα22GlcNAc(α)46Mannan23GlcNAc(β)47Xylan24GlcNAc(β1,3)GalNAc(α)48Dextran Open table in a new tab The volume of all solutions added to the glycan array was 10 μL/well, except where stated. Serum samples were diluted 1:40 in 0.15 mol/L Tris-HCl, pH 7.2, 0.085 mol/L Mg2SO4, 0.05% Tween-20 (TBST) containing 1% bovine serum albumin (BSA; Sigma), dispensed into the plates using a Tecan Genesis Workstation 200 automated handling system (Tecan, Zurich, Switzerland), and incubated for 60 minutes at 37°C. The plates were then washed with 250 μL/well phosphate-buffered saline (PBS) with .05% Tween-20 (PBST; Sigma) in an automatic plate washer (PowerWasher; Tecan, Zurich, Switzerland). The respective isotype-specific biotinylated goat anti-human Ig antibodies against IgG, IgA, or IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) were then added and incubated for 60 minutes at 37°C. Following washing with PBST, streptavidin-conjugated europium .1 μg/mL (Perkin-Elmer, Boston, MA) was added for 30-minute incubation at 37°C in the dark and washing with PBST. Delfia enhancement solution (Perkin-Elmer) was then added, and plates were incubated 30–45 minutes in the dark at room temperature. Fluorescence was read with a Victor 1420 multilabel counter (Perkin-Elmer) using time-resolved fluorescence settings of 340/612 nm (excitation/emission). To determine positive reactivities for the results obtained from the glycan array, we used relative fluorescence units (RFU). The cut-off values were 2.5 × 106 RFU for IgG and 0.74 × 106 RFU for IgA. These cut-off values were determined using receiver operating characteristic (ROC) curves (data not shown) to provide optimal positive and negative predictive values for differentiation between CD and UC patients for the relevant antibodies. After profiling antiglycan antibodies by the glycan array using indirect immunofluorescence, we established an enzyme-linked immunosorbent assay (ELISA) for the determination of the 3 most discriminative antibodies, thus facilitating wide-scale screening. Briefly, mannan and p-nitrophenyl derivatives of chitobioside and laminaribioside (the 3 glycans toward which most prominent and discriminative seroreactivities were detected in the discovery phase) were covalently attached to the surface of a 96-well microtiter plate via a linker as previously described.25Schwarz M. Spector L. Gargir A. Shtevi A. Gortler M. Altstock R.T. Dukler A.A. Dotan N. A new kind of carbohydrate array, its use for profiling antiglycan antibodies, and the discovery of a novel human cellulose-binding antibody.Glycobiology. 2003; 13: 749-754Crossref PubMed Scopus (127) Google Scholar Serum samples were diluted 1:400 in 5% DD8/1%BSA/TBST, pH 7.4, dispensed into the wells (50 μL per well), incubated for 30 minutes at 25°C, then washed with PBST buffer. Bound antibodies were labeled (30 minutes at 25°C) with 50 μL of either horseradish peroxidase (HRP)-conjugated goat anti-human IgA (1:2500) or IgG (1:5000) type-specific antibody (both antibodies from Jackson, ImmunoResearch Laboratories, West Grove, PA) and washed with PBST buffer, and 50 μL 3,3′,5,5′-tetramethylbenzidine (TMB) was added for detection. The optical density (OD) at 595 nm was read after 6 minutes with a Victor 1420 plate reader (Wallac, Turku, Finland); the enzymatic reaction was stopped with 50 μL TMB “stop” solution and read at 450 nm. Serum samples were considered as positive for IgA antichitobioside (ACCA), IgG antilaminaribioside (ALCA), and IgG anticovalently attached mannan (gASCA) if OD values at 450 nm were above the following cut-off values: .43, 1.55, and .4, respectively. These cut-off values were determined using ROC curves to provide 97%, 100%, and 92% specificity for ACCA, ALCA, and gASCA, respectively, for differentiation of CD and UC patients. Serum levels of ASCA were determined by ELISA using the QUANTA LiteTM ASCA IgG kit according to the manufacturer’s protocol (Inova Diagnostics Inc., San Diego, CA). The results were presented as arbitrary ELISA units (EU) that were calculated by dividing the average optical density of the sample by the average optical density of the ASCA IgG ELISA low positive. The result was multiplied by the number of units assigned to the ASCA IgG ELISA low positive, and the results were expressed as negative (<25 EU) or positive (>25 EU). Descriptive statistics were used for population and subgroup characteristics. For results obtained using the glycan array, ROC curves (generated by plotting sensitivity vs [1−specificity]) were calculated for the GlycoChip data. The curves were used to determine cut-off values providing optimal positive and negative predictive values. For comparison between the study groups, and testing association with clinical parameters, OD results were transformed using log10 for achieving distributions as close as possible to normal distribution. We used Student t test to assess significance differences in antiglycan antibodies between groups and χ2 test for nonparametric variables. Pearson correlation was used to test association between antibody levels and age or gender; a P value < .05 was considered to be statistically significant. To evaluate the association between disease phenotypes and the level of immune response toward the 3 glycan antigens, laminaribioside, chitobioside, and oligomannan, simultaneously, we performed quartile sum analysis.17Landers C.J. Cohavy O. Misra R. Yang H. Lin Y.C. Braun J. Targan S.R. Selected loss of tolerance evidenced by Crohn’s disease-associated immune responses to auto- and microbial antigens.Gastroenterology. 2002; 123: 689-699Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar For each antigen, patients whose antibody levels were in the first, second, third, and fourth quartile of the distribution were assigned a quartile score of 1, 2, 3, and 4, respectively. By adding individual quartile scores for each glycan antigen, a quartile sum score (range, 3–12) was obtained to represent the cumulative quantitative immune response toward all 3 antigens for each patient.18Mow W.S. Vasiliauskas E.A. Lin Y.C. Fleshner P.R. Papadakis K.A. Taylor K.D. Landers C.J. Abreu-Martin M.T. Rotter J.I. Yang H. Targan S.R. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.Gastroenterology. 2004; 126: 414-424Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar The Cochran-Armitage test for trend was used to test whether there was a linear trend in the proportion of patients with a disease phenotype characteristic as the level of antibody responses increased by quartiles. A P value (P trend) < .05 suggests that the linear trend is statistically significant. Serum samples were obtained from patients with CD (n = 72), UC (n = 56), and healthy controls (n = 41). There were no significant differences in demographic parameters between the groups studied (Table 1). As expected, bowel operations and use of biologic treatments (Infliximab) were significantly more frequent in the CD than the UC group (P < .0001 and P = .049, respectively), whereas the use of 5-aminosalicylic acid (5-ASA) was significantly higher in the UC group (P = .0001). Serum samples were initially screened for 48 different glycans using the GlycoChip glycan array (Table 3). In addition to antibodies against mannan and mannan residues, 1 IgG and 1 IgA antiglycan antibodies were significantly elevated in the sera of CD patients compared with UC patients (P < .001) (Table 4, Table 5). Anti-Glc(β1,3)Glc(β) (laminaribioside) IgG and anti-GlcNAc(β1,4)GlcNAc(β) (chitobioside) IgA were the most prominent antibodies, demonstrating the highest mean RFU in the CD group, and the most significant P values for discrimination between CD and UC patients. Anti-Glc(β1,3)Glc(β) (laminaribioside) IgG was also significantly higher in CD patients compared with healthy controls (P = .05), whereas anti-GlcNAc(β1,4)GlcNAc(β) (chitobioside) IgA was comparable between CD patients and healthy controls (P = .15). These novel antibodies were thus des" @default.
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• W2030421888 title "Antibodies Against Laminaribioside and Chitobioside Are Novel Serologic Markers in Crohn’s Disease" @default.
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