Autoantibodies against exocrine pancreas in Crohn's disease are directed against two antigens: The glycoproteins CUZD1 and GP2  Lars Komorowski, Bianca Teegen, Christian Probst, Karola Aulinger-Stöcker, Christian Sina, Klaus Fellermann, Winfried Stöcker  Journal of Crohn's and Colitis  Volume 7, Issue 10, Pages 780-790 (November 2013) DOI: 10.1016/j.crohns.2012.10.011 Copyright © 2012 European Crohn's and Colitis Organisation Terms and Conditions

Figure 1 Binding of pancreatic autoantigens to lectins using a glycoprotein-capture ELISA (A): streptavidin-coated microplates were used to immobilize biotinylated lectins (UEA I, WGA, DBA, SBA, RCA, PNA, or ConA). The solid phases were then incubated, subsequently with cell-free pancreas lysate, human sera from patients with Crohn's disease, anti-human IgG HRP conjugate, and tetramethylbenzidine substrate. Photometry was carried out at 450nm after the addition of 1volume1mol/L H2SO4. For each serum sample, the OD values of a second incubation omitting the cell-free pancreas lysate were subtracted. Results of representative incubations. (B) Coomassie-stained SDS-PAGE of the UEA I-purified pancreas fraction. Lane 1: molecular mass markers; kDa indicated; lane 2: 4μg UEA I-purified pancreas fraction. Journal of Crohn's and Colitis 2013 7, 780-790DOI: (10.1016/j.crohns.2012.10.011) Copyright © 2012 European Crohn's and Colitis Organisation Terms and Conditions

Figure 2 ELISA reactivity of the UEA I-purified pancreas fraction. Microplates were used to immobilize the UEA I-purified pancreas fraction. The solid phase was then incubated, subsequently with human sera, anti-human IgG HRP conjugate, and tetramethylbenzidine substrate. Photometry was carried out at 450nm after the addition of 1volume1mol/L H2SO4. Sera from 96 Crohn's disease patients, 39 ulcerative colitis patients, and from 50 healthy blood donors were analyzed. Journal of Crohn's and Colitis 2013 7, 780-790DOI: (10.1016/j.crohns.2012.10.011) Copyright © 2012 European Crohn's and Colitis Organisation Terms and Conditions

Figure 3 Immunofluorescence double staining of human pancreas and transfected HEK293: frozen sections of human pancreas and HEK293 transfected with either CUZD1 or GP2 were co-incubated with 1:100 diluted human serum and monoclonal antibodies. Bound antibodies were visualized with anti-human IgG Cy3 and anti-mouse IgG Cy2 conjugate. (A) PAb of the reticulogranular pattern versus 0.8μg/mL monoclonal antibody against CUZD1; (B) PAb of the droplet pattern versus 0.8μg/mL monoclonal antibody against GP2. (C) Frozen sections of human pancreas were co-incubated with 0.8μg/mL Cy3-labeled anti-CUZD1 and 0.8μg/mL Cy2-labeled anti-GP2 monoclonal antibodies. The scale bar represents a width of 20μm. Journal of Crohn's and Colitis 2013 7, 780-790DOI: (10.1016/j.crohns.2012.10.011) Copyright © 2012 European Crohn's and Colitis Organisation Terms and Conditions

Figure 4 Antigen capture ELISA for the determination of human IgG antibodies. Microplates were used to immobilize monoclonal antibodies against either CUZD1 or GP2. The solid phase was then incubated, subsequently with the UEA I-purified pancreas fraction, human sera, anti-human IgG HRP conjugate, and tetramethylbenzidine substrate. The sera from 96 Crohn's disease patients, 39 ulcerative colitis patients, and from 50 healthy blood donors were analyzed. (A) Indirect ELISA as in Fig. 2 versus CUZD1 capture ELISA; (B) indirect ELISA as in Fig. 2 versus GP2 capture ELISA. None of the healthy blood donor sera exhibited reactions above the cut-off limits of each test system. Journal of Crohn's and Colitis 2013 7, 780-790DOI: (10.1016/j.crohns.2012.10.011) Copyright © 2012 European Crohn's and Colitis Organisation Terms and Conditions