title: Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against "rings and rods"-associated inosine-5'-monophosphate dehydrogenase 2 in viral hepatitis C.
abstract: OBJECTIVE
  Autoantibodies against so-called "rings and rods" structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories.
  
  
  METHODS
  CTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-"rings and rods" antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors.
  
  
  RESULTS
  We found that all sera with anti-"rings and rods" reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2.
  
  
  CONCLUSIONS
  We conclude, that IMPDH2 is indeed the main target of anti-"rings and rods" while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen.
authors: Institute of Experimental Immunology, EUROIMMUN AG, Lübeck, Germany.
authors: Christian
authors: C
authors: Probst
authors: Christiane
authors: C
authors: Radzimski
authors: Inga Madeleine
authors: IM
authors: Blöcker
authors: Bianca
authors: B
authors: Teegen
authors: Dimitrios P
authors: DP
authors: Bogdanos
authors: Winfried
authors: W
authors: Stöcker
authors: Lars
authors: L
authors: Komorowski
mesh_terms: D000328:Adult; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000032:analysis* / Q000097:blood / Q000276:immunology*; D019731:Carbon-Nitrogen Ligases / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D057809:HEK293 Cells; D006526:Hepatitis C / Q000097:blood / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007168:IMP Dehydrogenase / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000097:blood / Q000276:immunology / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D011994:Recombinant Proteins; D007168:IMP Dehydrogenase; C501687:IMPDH2 protein, human; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase