Possible Use Of P20 Antigen In Serodiagnosis Of Inflammatory Bowel Disease

Crohn\u27s disease (CD) is an idiopathic, chronic, relapsing inflammatory disorder, which is most commonly involved terminal ileum and colon. The incidence and prevalence of CD has dramatically increased during the last 50 years; however, the etiology and mechanism of this disorder remain unveiled. Besides genetic susceptibility, recent integrated researches investigated the role of environmental triggers such as microflora, measles viruses and mycobacteria in the pathogenesis of CD. The association between M. avium subsp paratuberculosis (MAP) and CD has been heightened because of clinical resemblance to Johne\u27s disease (JD), a granulomatous enteritis in ruminants caused by MAP. Isolation of MAP from tissue and milk samples from CD patients and from commercial pasteurized milk and dairy products from JD-infected animals implies a possible re-classification of CD as zoonotic disorder. Clinical signs and symptoms of CD are often non-specific and a challenge to distinguish it from other disorders. Current methods for inflammatory bowel disease diagnosis, especially for CD are highly invasive, distressing and expensive. In this study, the recombinant clone pB11 containing 1.1 kb insert, identified from a MAP genomic library constructed in E. coli, expressed a 20 kDa (p20) antigen encoded on 549 bp partial MAP gene with an ORF cloned in frame within pBAD/His cloning vector. Immunoreactivity of p20 was confirmed by Immunoblot. Purified p20 antigen was then used in the development of an enzyme-linked immunosorbent assay (ELISA) for possible serodiagnosis of Inflammatory Bowel Disease (IBD) associated with MAP infection. All variables associated with ELISA test with regard to concentrations, washes and incubations were optimized using hyper immune rabbit t-anti-MAP polyclonal IgG antibodies and sera from CD and non-CD subjects. The cut-off value for positive response was established as 0.3 following the analysis of statistically formulated samples from normal and non-CD subjects. The developed ELISA test was then used to test a blindly coded 2 17 clinical sera. All sera samples were tested in duplicates and in both p20-coated and uncoated micro titer plates. Consequently, 116/134 (87%) CD sera were positive compared to 24/83 (33%) non-CD sera (P\u3c0.05). Specifically anti-MAP IgG was detected in 8/22 (36%) Ulcerative colitis and 16/61 (26%) non-IBD sera. p20-ORF encoding sequence was recloned (pB11/B6) and the expressed protein reactivity remained consistent. Moreover, the full length of the cloned gene was also identified through blast and alignment analysis and predicted to encode 346 amino acids with unknown function and no identity with other known proteins. The latter supports the clinical data, which reflect on the unique characteristics of this antigen. The result so far suggests that the recombinant clone and its subclone derivative may have potential role in serodiagnosis of C