科研之友
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Background: Indirect immunofluorescence (HET) on in house HEp-2 cell preparations revealed a novel antibody giving a granular cytoplasmic pattern not described before, which on two commercial cell preparations revealed a "rings and rods" pattern. This pattern was also observed in four HCV-RNA carriers and prompted the identification of the reactive antigen and the evaluation of the antibody prevalence in HCV-RNA carriers and control groups.
Methods: The antigen's molecular weight was determined by radioimmunoprecipitation of (35)S-methionine labeled cell proteins. Expression library screening and sequencing was performed by standard techniques using an oligo(dT)-primed human HeLa cell cDNA expression library. Antibodies against the novel antigen Inositol-5'-monophosphatdehydrogenase 2 (IMPDH2) were analyzed by HET, western blot, line blot, and radioimmunoprecipitation assay (RIPA). HET was performed on commercial HEp-2 cells and cells cultivated in house for 24 60 hours, with or without the IMPDH2 inhibitors mycophenolic acid (MPA) or ribavirin, and subjected to various fixation conditions. Western and line blots were performed with IMPDH2 synthesized in E. coli, RIPA with (35)S-methionine-IMPDH2 from in vitro transcription/translation products. Sera screened were positive for HCV-RNA (108), HBV-DNA (100), anti-mitochondrial (31), anti-actin (42), and anti-nuclear antibodies (51) and negative for HCV-RNA (100) and blood donors (100).
Results: IMPDH2 is capable of considerable intracellular rearrangements (upon action of inhibitors like MPA and ribavirin), which explains the contrasting immunofluorescence patterns in cells from different sources. By RIPA, proven to be the sole assay suitable for screening of anti-IMPDH2 in human sera, autoantibodies were found in 35.2 % of HCV-RNA carriers and in low concentrations in 3.1 % of anti-actin positive patients suspicious of autoimmune hepatitis. Antibodies reacted preferentially with conformational epitopes. Compared to the low concentration of anti-IMPDH2 found in other disease groups, high antibody concentrations were observed in HCV-RNA carriers.
Conclusions: The common occurrence of anti-IMPDH2 in HCV-RNA carriers may be related to ribavirin therapy, causing intracellular aggregation of IMPDH2 thereby altering its immunogenicity. In this study the "rods and rings" immunotborescence pattern observed could be ascribed to anti-IMPDH2. Anti-IMPDH2 may cause difficulties in interpretation of immunofluorescence patterns in routine autoantibody testing. (Clin. Lab. 2011;57:753-765)
- 出版日期2011
