title: Autoantibodies against exocrine pancreas in Crohn's disease are directed against two antigens: the glycoproteins CUZD1 and GP2.
abstract: BACKGROUND
  Autoantibodies against exocrine pancreas (PAb) have been reported to be pathognomonic markers of Crohn's disease (CD). Recently, the glycoprotein GP2 has been proposed as the exclusive target for PAb but two equally prevalent binding patterns can be observed in the indirect immunofluorescence test (IIFT) using cryosections of human pancreas: a reticulogranular and a droplet pattern.
  
  
  OBJECTIVE
  To identify autoantigens corresponding to the staining patterns.
  
  
  METHODS
  Different lectins were screened for their ability to immobilize PAb-reactive glycoproteins from cell free human pancreas. The glycoproteins were then purified via UEA-I affinity chromatography and identified by mass spectrometry. The two candidate autoantigens were separately expressed in HEK293 cells, and the recombinant cells applied as substrates in IIFT to analyze sera from 96 patients with CD, 89 controls and hybridoma supernatants during the generation of murine monoclonal antibodies.
  
  
  RESULTS
  The UEA-I eluate was able to neutralize PAb reactivity of both patterns in IIFT. It contained two major constituents which were identified as the glycoproteins CUZD1 and GP2. With the recombinant cells, 35.4% of the CD patients exhibited positive reactions (CUZD1 alone 19.8%, GP2 alone 9.4%, and both antigens 6.2%). The reaction with the CUZD1 expressing cells was strictly correlated to the reticulogranular pattern, whereas the antibodies causing the droplet pattern stained the GP2 expressing cells. Antigen-capture ELISA using the newly generated monoclonal antibodies against CUZD1 and GP2 verified this relationship.
  
  
  CONCLUSIONS
  The concordant reactivities of the different platforms can be regarded as a proof for the authenticity of the two identified autoantigens.
authors.affiliation: Institute of Experimental Immunology, Euroimmun AG, Luebeck, Germany. Electronic address: l.komorowski@euroimmun.de.
authors.forename: Lars
authors.initials: L
authors.lastname: Komorowski
authors.forename: Bianca
authors.initials: B
authors.lastname: Teegen
authors.forename: Christian
authors.initials: C
authors.lastname: Probst
authors.forename: Karola
authors.initials: K
authors.lastname: Aulinger-Stöcker
authors.forename: Christian
authors.initials: C
authors.lastname: Sina
authors.forename: Klaus
authors.initials: K
authors.lastname: Fellermann
authors.forename: Winfried
authors.initials: W
authors.lastname: Stöcker
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D003093:Colitis, Ulcerative / Q000097:blood; D003424:Crohn Disease / Q000097:blood*; D005260:Female; D058851:GPI-Linked Proteins / Q000276:immunology* / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008875:Middle Aged; D046790:Pancreas, Exocrine / Q000276:immunology*; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; C488236:CUZD1 protein, human; C489339:GP2 protein, human; D058851:GPI-Linked Proteins; D008565:Membrane Proteins
keywords: AP; AUC; Autoantibodies;; CD; CUZD1;; Crohn's disease; Crohn's disease;; Exocrine pancreas;; FITC; GP2; GPI; HRP; IBD; IIFT; MALDI-TOF; PAGE; QIT; ROC; SDS; UC; UEA-I; Ulex europaeus agglutinin I; alkaline phosphatase; area under the curve; fluorescein isothiocyanate; glycosylphosphatidylinositol; horse radish peroxidase; indirect immunofluorescence test; inflammatory bowel disease; matrix-assisted laser desorption/ionization trap-time of flight mass spectrometry; polyacrylamide gel electrophoresis; quadrupole ion trap; receiver-operating characteristics; sodium dodecylsulfate; ulcerative colitis