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O073 - A CELL BASED ASSAY FOR DETECTION OF ANTI-FIBRILLARIN AUTOANTIBODIES (ID 75)
Abstract
Background and Aims
Anti-fibrillarin(U3-RNP) antibodies are useful in establishing diagnosis and prognosis in Systemic Sclerosis(SSc). Anti-fibrillarin causes a characteristic clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2-IFA). Immunoprecipitation is the gold standard for anti-fibrillarin determination and solid-phase immunoassays (SPIA) yield inconsistent results, possibly because of antigen denaturation. We established an anti-fibrillarin Cell-Based Assay (CBA) and compared its performance with immunoprecipitation and SPIA.
Methods
To the fibrillarin gene, we added a TransMembrane Signal(TMS) that “relocates” fibrillarin to the cytoplasmic membrane. HEp-2 cells transfected with the plasmid pCMV_TMS-fibrillarin_P2A_OFP-myc were used as substrate for IFA in the CBA. Sixty-two samples with high-titer nucleolar pattern by HEp-2-IFA (41 AC-9/clumpy and 21 AC-8/AC-10 homogeneous/punctate) were tested in the fibrillarin-CBA, immunoprecipitation, line-blot and ELISA.
Results
TMS-fibrillarin was properly relocated to the cytoplasmic membrane and recognized by autoantibodies (Figure). Thirty-eight of 41 AC-9/clumpy nucleolar samples (92.7%) and none of 21 samples with other nucleolar patterns were positive for anti-fibrillarin in CBA. There was 100% agreement between the positive/negative results in the CBA and the immunoprecipitation tests. Among the 38 CBA-positive samples, only 15(39.5%) and 11(29%) were considered positive in the line-blot and ELISA assays, respectively. Among the 24 CBA-negative, only 1 was positive by line-blot and 1 by ELISA.
Conclusions
With an innovative strategy of relocating the autoantigen to the cell membrane, we developed a new assay for detection of anti-fibrillarin autoantibodies. This new CBA presented high sensitivity and specificity comparable to the gold standard Immunoprecipitation. Additionally, the fibrillarin-CBA showed much higher sensitivity than SPIA such as line-blot and ELISA.