Anti-neutrophil cytoplasmic antibody: Anti-neutrophil cytoplasmic antibody (ANCA) is classified according to two staining patterns: cytoplasmic ANCA (cANCA), in which the entire cytoplasm is stained, and perinuclear ANCA (pANCA), in which the area around the nucleus is stained. cANCA is expressed in Wegener’s granulomatosis and other diseases, and pANCA is observed in IBD. In IBD, the antigen that corresponds to pANCA is thought to be histone 1, whereas in vasculitis, it is thought to be proteinase 3 and myeloperoxidase. pANCA is regarded as an autoantibody that is induced by a cross-reaction with intestinal bacterial antigens. pANCA is detected in 60%-70% of UC cases, 10%-15% of CD cases, and less than 5% of non-IBD colitis cases[1,2]. Moreover, patients with pANCA-positive CD exhibit a clinical phenotype resembling that of UC[3]. Unlike the pANCA in vasculitis, IgG from pANCA-positive UC was not able to activate a neutrophil respiratory burst[4].

Pancreatic antibody: Pancreatic antibody (PAB) is an antibody to a trypsin-sensitive protein in pancreatic secretions[5]. PAB is positive in 20%-40% of CD cases and 5% of UC cases[6]. PAB expression may exhibit racial differences. One study describes a positive rate of 46% among Chinese patients with CD, compared with 22% among Western patients[7]. The role of PAB in the diagnosis of IBD requires further study.

Anti-Saccharomyces cerevisiae antibody: Anti-Saccharomyces cerevisiae antibody (ASCA), an anti-glycan antibody, is an antibody against mannan on the cell wall surface of baker’s yeast (S. cerevisiae). The fact that CD is common in bakery workers (baker’s disease) and that the disease activity of CD decreases in response to a diet low in baker’s yeast suggest that ASCA is involved in the pathogenesis of CD. On the other hand, because mannan, which is the antigen recognized by ASCA, is a cell wall constituent of living organisms, including baker’s yeast, and is also present as a protein in the human body, the immunogen may be something other than the yeast. Increases in IgG and IgA ASCAs have been reported in patients with CD, and although IgA ASCA has a high specificity, its sensitivity is not so high; instead, IgG ASCA is generally used as a biomarker[8]. The IgG ASCA-positive rate is 60%-70% in patients with CD, 10%-15% in patients with UC, and less than 5% in patients with non-IBD colitis[2,8]. ASCA also increases significantly in Japanese patients with CD, but the titers and positive rates are lower than those in Western CD patients, indicating that the expression of ASCA may also be affected by race[9].

Novel anti-glycan antibodies: New anti-glycan antibodies, including IgG anti-laminaribioside carbohydrate antibody (ALCA), IgA anti-chitobioside carbohydrate antibody (ACCA), and IgG anti-mannobioside carbohydrate antibody (AMCA), were reportedly elevated in the sera of CD patients in recent studies using GlycoChip microarrays[10,11]. These antibodies mainly target the cell wall components of microorganisms, including yeasts, fungi, and bacteria. The positive rate of both ALCA and ACCA is 20%-40% in CD and 10% in UC, but an improvement in CD diagnostic utility is expected using a combination of these new antibodies with ASCA. Laminaribioside, a laminarin component, is known to promote dectin-1-dependent cytokine production from macrophages and T-cell proliferation, and the increased expression of chitinase 3-like-1, which has the ability to bind chitin (containing chitobioside as a component), has been reported in intestinal epithelial cells and lymphocytes at sites of IBD. Both of these findings are very interesting and suggest that these cell wall components are involved in the pathogenesis of IBD.

Anti-outer-membrane porin C antibody: The anti-outer-membrane porin C (OmpC) antibody is an antibody to an outer-membrane protein of Escherichia coli. IgA anti-OmpC is positive in 55% of CD cases, 5%-10% of UC cases, and 5% of non-IBD colitis cases, and is even positive in 17%-36% of indeterminate colitis cases[12-16].

Anti-Cbir1 antibody (anti-flagellin antibody): Anti-flagellin (Cbir1) antibody is an antibody to a flagella component of indigenous bacteria in colitis mouse model[17]. Cbir1 has been cloned from the sera of colitis mice as a colitis-associated bacterial antigen. IgG anti-Cbir1 is positive in 55% of CD cases, 10% of UC cases, and 8% of non-IBD colitis cases[17-19]. After binding to Toll-like receptor-5, flagellin activates NF-κB and promotes the production of pro-inflammatory cytokines. Notably, colitis developed when flagellin-specific CD4⁺Th1 cells were transferred into severe combined immunodeficiency mice, suggesting that the antigen is involved in the pathogenesis of colitis[18].

Anti-I2 antibody: Anti-I2 antibody is an antibody against Pseudomonas fluorescens component I2 isolated from mononuclear cells in the intestinal mucosa of patients with CD. IgA anti-I2 is positive in 55% of CD cases, 10% of UC cases, and 20% of non-IBD colitis cases[12,15,16]. Importantly, I2 is active as a T-cell superantigen.

Anti-Mycobacterium avium subspecies paratuberculosis antibody:Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease, an intestinal infectious disease in ruminants and primates, in which non-caseating granulomas develop in the terminal ileum and colon. Based on the similarity to the pathohistological findings in CD and the fact that MAP has been isolated and cultured from intestinal tissue and breast milk in CD patients, MAP is thought to be involved in the pathogenesis of CD[20]. Naser et al[21] have reported increased titers of antibodies to MAP-specific proteins (p35 and p36) in the sera of CD patients. Nakase et al[22] prepared a recombinant protein produced by IS900 and observed elevated titers in CD patients when the recombinant protein was used as an antigen to measure the anti-IS900 level using an ELISA. However, MAP has been isolated from healthy subjects as well, and whether MAP itself has the ability to infect humans remains unknown.

Anti-Caenorhabditis elegans antibody: Oshitani et al[23] analyzed HLA-DR-binding antigen peptides in the intestinal mucosa of IBD patients and discovered an antigen expressed by the nematode Caenorhabditis elegans (C. elegans), which lives in the soil. Subsequently, they reported an increase in anti-C. elegans antibody titers in the sera of patients with CD or UC[24].

Anti-cocktail multiple antigenic peptide antibody: Saito et al[25] discovered 4 different peptides (cocktail multiple antigenic peptides, cocktail MAP) that reacted with the sera of CD patients by screening with a peptide phage library and established an ELISA that detects the anti-cocktail MAP antibody. The rate of positivity was 44% among CD patients, but positivity was rare among UC patients and healthy subjects. No reports of antigens corresponding to these peptides have been found.

Anti-TCP antibody: We used IgG in sera from CD patients to screen a T7 phage display library produced using colon cancer cDNA[26]. We selected a phage that specifically bound to a high percentage of sera samples from CD patients and then determined the amino acid sequence of the expressed peptide[2]. A previously unidentified, novel peptide (TCP peptide) was found, and an ELISA produced by converting the TCP peptide into a solid phase showed that while 61.7% of the CD patients who were examined exhibited positive seroreactivity, a positive result was less common among patients with UC (7.3%), non-IBD colitis (0%), colon cancer (11.4%), or healthy subjects (2.8%). These results demonstrated that an antibody to the TCP peptide is present in a high percentage of sera samples from CD patients.

Interestingly, when mononuclear cells are stimulated with the TCP peptide, they produce large amounts of pro-inflammatory cytokines, suggesting that antigens associated with this peptide are highly involved in the pathogenesis of CD. Homology has been found between the TCP peptide and rice- and microorganism-derived proteins, but no specific antigen has yet been identified. The major differences from the anti-cocktail MAP antibody described above[25] are the combination of four different, branched peptides in the cocktail MAP, whereas only a single, linear peptide is present in the TCP peptide.

At present, no single ideal serological antibody exists for the diagnosis of IBD, and the use of combinations of several markers has been attempted. In patients already diagnosed as having CD or UC, the predictive value of a pANCA-negative/ASCA-positive result was 95% for CD, while that of a pANCA-positive/ASCA-negative result was 90% for UC[1,27].

In patients with indeterminate colitis, the combination of pANCA and ASCA was useful for predicting whether the disease would progress to either CD or UC. A prospective study showed that the predictive value of this combination was 80% for progression to CD in pANCA-negative/ASCA-positive patients and 63.6% for progression to UC in pANCA-positive/ASCA-negative patients. Moreover, approximately 50% of the indeterminate colitis patients were pANCA-negative/ASCA-negative[28]. A poor postoperative outcome following ileal pouch-anal anastomosis surgery has also been reported in a patient with indeterminate colitis who progressed to CD[29].

Reportedly, 44% of CD patients who are negative for ASCA are ALCA-positive or ACCA-positive[10,11], and approximately 40% of CD patients who are negative for pANCA, ASCA, anti-OmpC, and anti-I2 are anti-Cbir1-positive[18]. These data indicate that the combination of several antibodies could further improve the diagnostic performance for CD.

Israeli et al[30] assessed the availability of ASCA to predict the development of CD. During a 3-year observation of healthy Israeli soldiers who had enlisted in the army, 32 developed CD, and 10 (31.3%) of these soldiers were ASCA-positive at the time of enlistment. On the other hand, none of the 8 soldiers who developed UC were ASCA-positive[30]. Moreover, the fact that familial clustering is seen in the expression of ASCA and anti-OmpC in patients with CD and in the expression of pANCA in patients with UC and CD[31-34] suggests that a genetic predisposition is involved in the expression of these antibodies.

The titers and numbers of microbial antibodies are closely associated with the clinical phenotypes of CD, including small bowel disease, intestinal complications (such as stricturing and internal penetrating disease behaviors), and the indications for small bowel surgery. Small bowel disease, a stricturing-type phenotype, a penetrating-type phenotype, and the need for small bowel surgery are common among ASCA-positive patients; a penetrating-type phenotype is common among anti-OmpC-positive patients; small bowel disease, a stricturing-type phenotype, and a penetrating-type phenotype are common among anti-Cbir1-positive patients; and a stricturing-type phenotype and the need for small bowel surgery are common among anti-I2-positive patients. Moreover, patients who were triple positive for ASCA, anti-OmpC and anti-I2 were more likely to have undergone small bowel surgery than seronegative patients (OR = 8.6, P < 0.001)[35,36]. Similar results were obtained in a multicenter prospective study of pediatric patients with CD that utilized four antibodies: ASCA, anti-OmpC, anti-Cbir1 and anti-I2[37]. Thus, the greater the number of positive antibodies, the more likely that the stricture and penetration will develop as complications; of note, when all four antibodies are positive, the frequencies of these complications are 11 times higher than when all four antibodies are negative. Accelerated progression to a penetrating and/or stricturing disease has also been shown in patients in whom at least one antibody was positive, compared with seronegative patients. Furthermore, in a study that added novel anti-glycan antibodies, samples with higher titers of each antibody (ASCA, anti-OmpC, ALCA, ACCA and AMCA) were associated with more severe and complex complications (stricture and penetration) and a higher rate of surgery[14]. On the other hand, a need for early surgery and frequent complications associated with postoperative ileal pouchitis have been found in pANCA-positive UC cases[38,39].

In an autoantibody study, the frequencies of penetration, anal lesions and extra-intestinal complications were elevated in PAB-positive CD cases[5]. Autoantibodies to extra-intestinal components, including the pancreas, phospholipids, tissue plasminogen activator and sperm, which were reported in a minority of IBD patients, could conceivably contribute to the pathogenesis of some extra-intestinal complications, such as pancreatitis, thromboembolism, and infertility[40].

It would be of great clinical and economic significance if clinicians could utilize serological antibody measurements to predict the response to treatment and to implement individualized treatment plans based on this information. This goal is particularly important because recent biologics are costly and can be associated with serious adverse events. Infliximab, an anti-tumor necrosis factor-α agent, has been reported to have little efficacy in pANCA-positive CD patients[41], and a subsequent study demonstrated a similar result in pANCA-positive/ASCA-negative CD patients[42]. Infliximab also has little efficacy in pANCA-positive/ASCA-negative UC patients[43]. Also, CD patients who responded to antibiotics were positive for anti-I2 or anti-OmpC, which are antibodies against intestinal bacteria[14]. Furthermore, pANCA-positive UC has been reported to be resistant to medical treatments[44].