Background: Crohn's Disease (CD), a chronic inflammatory bowel disease (IBD), is thought to be multifactorial, involving an interaction between genetic susceptibility, undefined environmental triggers, and immune-mediated tissue injury. Biochemical and other molecular approaches identified isolates from intestinal tissues of patients with CD as Mycobacterium avium subsp paratuberculosis (MAP), the causative agent of Johne's disease, a granulomatous bowel disease in ruminants similar to CD. MAP has been identified directly in resected tissues of increasing numbers of CD patients at a frequency significantly higher than those of controls. Treatment of CD patients, which depends on the location and severity of disease, complication, and response to previous treatment is most often to control the disease. There is no cure. Diagnosis of this disease requires a series of tests including upper gastrointestinal endoscopy and colonoscopy. These tests are expensive, inconvenient and require hospitalization. Objective: A blood serologic test is sought for diagnosis of CD patients infected with MAP. Methods: The recombinant E. coli clone pBl 1 containing a 1,302 bp MAP DNA insert and expressing a 20 kD protein has been grown, induced by arabinose and then harvested by centrifugation. Protein extracts were prepared, quantitated and then subjected to Isoelectricfocussing (IEF) in ampholyte buffer pH 3-10. Twenty fractions were collected, quantitated and then analyzed on SDS-P AGE by silver staining and Imrnuboblotting. The immunoblots were screened with anti-express IgG monoclonal antibodies. Fractions containing the semipurified 20 KD protein were analyzed by immnoblot against 85 sera specimens with 1:30 dilution (43 CD patients and 42 controls). Both IgG and IgA response in each patient was determined. Results: Of 20 fractions collected, fractions 5 and 6 with a PI ranging from 4.18 to 5.01 reacted with the anti-express IgG antibodies. p20 with a 20 kD molecular weight was confirmed. These fractions contained fewer proteins bands with p20 being dominant. Of 43 CD sera specimens, 74% contained IgG response and only 50% contained IgA response to p20. On the other hand, of 42 controls, only 17% contained IgG and. 50 % contained IgA response.against p20 antigen. Conclusion: p20 reacted with CD IgG sera with frequency much higher than control sera (74% versus 17%) indicating a great potential for using p20 as a reagent in a quantitative ELISA assay for specific diagnosis of CD patients. Additionally, the data add strong support to MAP role in CD pathogenesis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been suggested as a causative agent of Crohn's disease (CD). Despite a long-term debate to prove this possibility, the role of this bacterium in the pathogenesis of CD is still a subject of controversy. The objective of the study was to develop a serodiagnostic assay for the diagnosis of CD in humans. METHODS: In the present study, five different ELISA assays were accessed: 1) IDEXX, a commercially available kit for the diagnosis of Johne's disease in ruminants; 2) an in-house developed assay based on total MAP cytoplasmic proteins, and three other assays based on recombinant MAP recombinant antigens a) a 23 kDa antigen, pB11/B7, b) a 35 kDa antigen, P35 and c) a 36 kDa antigen, P36. The last three proteins were identified from an expression genomic library of MAP that was constructed in our laboratory. A total of 43 sera samples were analyzed in this study, which included 14 CD patients, 14 Ulcerative Colitis (UC) patients, and 13 non-inflammatory bowl disease (IBD) patients. lmmunoblot and silver stain analyses were performed to confirm protein identity and purity. ELISA was developed and used to analyze the level of anti-MAP lgG antibodies in sera from patients. RES UL TS: The rate of positive ELISA results is based on previously published interpretation criteria. ELISA results using the IDEXX kit showed 12/14 (85.7%) positive for CD as compared to 7/13 (53.8%) for non-lBD and 6/14 (42.9%) for uc.· 8/14 (57.1%) of the CD sera were positive with the ELISA results based on MAP cytoplasmic proteins compared with 6/13 (46.2%) of non-lBD and 10/14 (71.4%) of UC. Further analyzing the recombinant proteins, when two out of three assays were used 12/14 (85.7%) CD (P<0.05), 0/13 (0.0%) non-lBD, and 1/14 (7.7 %) UC were positive. Moreover, when all three recombinant proteins are utilized for analysis, the specificity of the test greatly increased, giving 13/14 (92.9%) positive for CD, 3/14 (21.4%) for UC and 2/14 (14.3%) for non-lBD. CONCLUSION: MAP recombinant proteins, pB11/B7, p35, and p36 showed a strong reactivity with diagnosed CD patients while excluding healthy individuals and other IBD patients. In addition, they served as a great tool to distinguish between CD and UC patients. A larger sample size needs to be tested, none the less this data strengthens the role of MAP in CD etiology and suggests a great potential for using the recombinant-based assays for diagnosis and treatment monitoring of patients with inflammatory bowel disease.