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. AIM: 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.
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. AIM: 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 CDpatients 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: Ralf J Ludwig; Karen Vanhoorelbeke; Frank Leypoldt; Ziya Kaya; Katja Bieber; Sandra M McLachlan; Lars Komorowski; Jie Luo; Otavio Cabral-Marques; Christoph M Hammers; Jon M Lindstrom; Peter Lamprecht; Andrea Fischer; Gabriela Riemekasten; Claudia Tersteeg; Peter Sondermann; Basil Rapoport; Klaus-Peter Wandinger; Christian Probst; Asmaa El Beidaq; Enno Schmidt; Alan Verkman; Rudolf A Manz; Falk Nimmerjahn Journal: Front Immunol Date: 2017-05-31 Impact factor: 7.561
Authors: Márta Kovács; Katalin Eszter Müller; Mária Papp; Péter László Lakatos; Mihály Csöndes; Gábor Veres Journal: World J Gastroenterol Date: 2014-05-07 Impact factor: 5.742
Authors: Christopher J Day; Elizabeth N Tran; Evgeny A Semchenko; Greg Tram; Lauren E Hartley-Tassell; Preston S K Ng; Rebecca M King; Rachel Ulanovsky; Sarah McAtamney; Michael A Apicella; Joe Tiralongo; Renato Morona; Victoria Korolik; Michael P Jennings Journal: Proc Natl Acad Sci U S A Date: 2015-12-16 Impact factor: 11.205
Authors: Christos Liaskos; Eirini I Rigopoulou; Timoklia Orfanidou; Dimitrios P Bogdanos; Christos N Papandreou Journal: Clin Dev Immunol Date: 2013-04-22