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

Background and aims: 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.